Seedless fruit producing plants

ABSTRACT

The present invention is directed to seedless fruit producing plants. The present invention also comprises methods for production of said plants and the use of nucleic acids encoding cyclin SDS like proteins for the production of seedless fruits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/304,228, filed Nov. 23, 2018, which is a National Stage Entry ofPCT/EP2017/062093, filed May 19, 2017, which claims priority to EPPatent Application No. 16171462.1, filed on May 26, 2016, thedisclosures of which are hereby incorporated by reference in theirentirety.

The present invention is directed to seedless fruit producing plants.The present invention also comprises methods for production of saidplants and the use of nucleic acids encoding cyclin SDS like proteinsfor the production of seedless fruits.

Most commercial seedless fruits have been developed from plants whosefruits normally contain numerous relatively large hard seeds distributedthroughout the flesh of the fruit. Seedless fruits are e.g. known forwatermelon, tomato, cucumber, eggplant, grapes, banana, citrus fruits,such as orange, lemon and lime. As consumption of seedless fruits isgenerally easier and more convenient, they are considered valuable.

Fruit development normally begins when one or more egg cells in theovular compartment of the flower are fertilized by sperm nuclei frompollen.

Seedless fruits can result from two different phenomena. In some casesfruit develops without fertilization of the ovule by pollen, aphenomenon known as parthenocarpy. In other cases seedless fruits occurafter pollination when seed (embryo and/or endosperm) growth isinhibited or the seed dies early, while the remainder of the fruitcontinues to grow (stenospermocarpy). In contrast to parthenocarpy,stenospermocarpy requires pollination for initiation of fruit growth.

Seedless orange fruits are an example for parthenocarpy. Some orangevarieties (e.g. Navel) do not produce viable pollen. They however can becross-pollinated with pollen from other varieties. In case only the malesterile variety is grown in an orchard, there will be no pollination andparthenocarp seedless fruits will be produced. Propagation of therespective orange trees is commonly done by cuttings followed bygrafting to another rootstock.

Seedless bananas are triploid. Although pollination in some cases can benormal and, the vast majority of fruits is seedless. This is explainedby the uneven sets of chromosomes (3×) leading to improper division ofchromosomes during meiosis and as a consequence to the production ofnon-viable pollen. Without fertilization, triploid bananas are also ableto set and develop seedless fruits. Even when pollination takes place,at most one in three hundred fruits comprises a few seeds. This may bedue to the triploid pollen being non-viable, for the reasons explained.Therefore, banana plants can in general be seen to be parthenocarpic.Banana plants are commonly propagated asexually from side shoots orsuckers at the base of the main stalk, which can be removed andreplanted to continue the cultivar. Growers also propagate bananas bymeans of tissue culture, in particular for producing disease freematerial.

Seedless cucumber, seedless squash and seedless eggplant are examplesfor crops which can produce seedless fruits without pollination(parthenocarpy), e.g. under conditions where pollination is impaired(e.g. low temperatures). Nevertheless, commercial quality fruit can beproduced under these conditions. All these crops however can produceseed bearing fruits upon pollination. Therefore, these crops arefacultative parthenocarpic. Propagation of the crops can be done byself- or cross pollination, in vitro propagation, and grafting.

From tomato mutants it is also known that they can produce seedlessfruits under conditions where normal pollination/fertilization isimpaired (e.g. under circumstances of low temperature). Thus, thesemutants are also facultative parthenocarpic. Mutants known for showingthis phenotype are pat, pat-2 and the pat-3/pat-4 system. The genesunderlying these mutations are not known and the pat-3/pat-4 systemseems to depend on multiple loci.

Parthenocarpy has also been introduced into several plant species bymeans of genetic modification. Expression of a bacterial tryptophanmonooxygenase (iaaM) conferring auxin synthesis under control of theovule and placenta specific DefH9 promoter did induce parthenocarpy incucumbers (Yin et al., 2006, Clular & molecular Biotech. Letters 11,279-290), eggplant (Acciarri et al., 2002, BMC Biotech. 2(4)), tomato(Rotino et al., 2005, BMC Biotech. 5(32)) and tobacco.

These transgenic plants demonstrate the importance of plant hormones inseed and fruit development. That seed and fruit development are besidesother factors strongly under control of several plant hormones is wellknown in the art. Parthenocarpy, including the logical consequence offruit's seedlessness, can also be induced e.g. by exogenous applicationof plant hormones, in particular auxin or gibberellin (Ruan et al.,Trends in Plant Sci. 17(11), 1360-1385).

Seedless watermelons produced by breeders are examples forstenospermocarp crops. Normal watermelon plants are diploid (2n).Seedless fruit producing watermelons are hybrids produced by crossing amale diploid (2n) watermelon plant with a female tetraploid (4n)watermelon plant. The resulting F1 hybrid seeds are triploid (3n).Induction of fruit setting in the F1 hybrid plants requires pollination.As the triploid (3n) F1 hybrid plants do not produce fertile pollen, socalled pollinator or polliniser plants have to be planted in the samefield. The pollinator plants are diploid (2n). Generally a ratio ofpollinator to hybrid plants of around 1 to 3 must be planted in a givenscheme for providing sufficient pollen for pollinating all the F1 hybridplants. The cross-pollination between the diploid (2n) pollinator andthe flowers of the female triploid (3n) hybrid plant induces fruit setand leads to the production of seedless triploid fruits on the triploidhybrid plant. The diploid (2n) and tetraploid (4n) parents of the F1hybrid each produce seed bearing fruits and can both be propagatedindependently from each other by self-pollination.

Seedless grapes can be produced from plants being either parthenocarp orstenospermocarp. The variety Black Corinth is parthenocarp, whereasSultanina is stenospermocarp. Vine plants are in general propagated bycuttings and successive grafting to another rootstock.

Irregularities in meiosis can be a factor leading to plants producingseedless fruit. An example for plants producing seedless fruits is givenin Zhang et al. (2012, Scientia Horticulture 140, 107-114), disclosingseedless watermelons. A male and female sterile (MFS) mutant wasobtained from the progeny of a F1-hybrid after irradiation of its seedswith gamma-rays. Pollen from the MFS mutant was not viable at all.Seedless fruits are produced by the MFS plants, when pollinated withpollen from male fertile plants. The MFS watermelon plant therefore canbe classified as being stenospermocarpic. Ovules were also nearlyentirely non-viable, as almost no seeds were produced uponcross-pollination of MFS mutants with pollen from different male fertileplants. Incomplete synapsis and abnormal separation of chromatids duringmeiosis were observed in the MFS mutant and seen to be the cause of maleand female sterility. The genes responsible for the effects present inthe MFS mutant have not been identified but it seems likely that thephenotype in the MFS mutant is due to a single recessive gene.

Pradillo et al. (2014, Frontiers in Plant Sci. 5, Article 23, do:10.3389/fpls.2014.00023) reviews the knowledge in the art about geneswhich are involved in homologous recombination during meiosis inArabidospsis.

Azumi et al. (2002, EMBO J. 21(12), 3081-3095) describe the isolation ofan Arabidopsis mutant having defects in synapsis and bivalent formationin male meiosis and similar defects, although to a lesser extent, infemale meiosis. The mutation was designated “Solo Dancers” (sds) andshown to originate from a single recessive gene. SDS mutants are malesterile and strongly impaired in female fertility. Plants homozygous forthe sds mutation are male sterile but at least to a minor extend femalefertile, which was demonstrated by cross-pollination of the sds mutantplant with pollen from male fertile plants. SDS mutants therefore aremale sterile and strongly impaired in female fertility. The sds gene wasidentified to belong to the cyclin type protein encoding genes and hasbeen demonstrated to interact with Arabidopsis CDKs, Cdc2a and Ccdc2bproteins. SDS however has been identified to be a new, beforehandunknown cyclin type protein. De Muyt et al. (2009, PLOS genet. 5(9)e1000654, doi: 10.1371/journal.pgen.1000654) confirms that the sdsArabidopsis mutant has a recombination defect in meiosis and suggestthat the defect is caused by a misallocation of another protein (AtDMC1)in the cells during meiosis.

From above discussion it is evident, that the factors determining ifplants produce seedless fruits are multiple in nature and can reside inseveral, e.g. morphologic, physiologic and/or genetic causes.

For producing seedless fruits in stenospermocarpic crops, a femaleflower part of a plant must be pollinated. The stenospermocarpic cropsgrown today are male sterile. As a consequence, besides the femaleplant, a different male fertile plant (pollinator or polliniser) has tobe grown in addition in the same field. As the area used for thepollinator plants is at the expense of the area which is available forthe seedless fruit producing female plants, the yield per area undercultivation is reduced. In general, the pollinator plants are normalplants which can also be self-pollinated. Fruits produced by pollinatorplants however do produce seeds. In watermelon, the pollinator plantsare normally diploid (2n), which upon self-pollination produce seededfruits, which may in some instances also be harvested and soldseparately (see WO2012069539). For commercial reasons these seededfruits from the pollinator plants must not be mixed with the seedlessfruits. Therefore, it has to be ensured, that seedless fruits and seededfruits are separated upon or after harvest, which may make machineharvesting difficult or impossible or require a further processing stepafter harvesting. Those additional precautions to be taken increase theinput costs in seedless fruit production. In addition, pollinator plantsare developed so that they flower and produce sufficient viable pollenat the same time the female plant flowers and its stigma can acceptpollen for the induction of fruit set. Thus, the pollinator plant has tofit with the female plant producing seedless fruit in respect toflowering and fertilisation time. If flowering time of the pollinatorpant and the respective female plant is not sufficiently synchronised,pollination will not take place or only take place in an unsufficientamount of cases. As a result fewer fruits are produced by thestenospermocarpic female plant. Furthermore, it is well known in the artthat climate conditions, like rain, heat etc., may influence pollenproduction of a polliniser plant differently than stigma fertility timeof the genotypic different female plant. Therefore, climate conditionscan also lead to asynchrony of fertility time of pollinator and femaleplant with the effect of lowering the yield.

Respective disadvantages are not applicable to the plants of theinvention described herein below.

It is, therefore, an object of the invention to overcome thedisadvantages of seedless fruit producing plants currently cultivated.

In a population of mutagenized M2 diploid watermelon plants a plantproducing seedless fruits was observed. The mutant plant was designatedEMB1. Surprisingly, pollen of said plant could be used to makeback-crosses. Thus, contrary to the seedless fruit producing plantsknown in the art, the plants disclosed herein are male fertile. Theback-crosses were self-pollinated and 25% of the plants obtainedtherefrom produced seedless fruits. A mutant allele (emb1) wasidentified which caused the seedless fruit phenotype, i.e. when diploidplants homozygous for emb1 (emb1/emb1) were either self-pollinated orpollinated by pollen from another plant, they produced seedless, diploidfruits. Thus, the seedless fruit phenotype occurs in plants beinghomozygous for a recessive mutation in the emb1 allele. The wild typeprotein encoded by the wild type allele corresponding to the mutant emb1allele of the invention has some similarities but also significantdifferences with cyclin SDS proteins and was, therefore, designated“cyclin SDS like protein”. The sequence identity between the cyclin SDSprotein and its encoding nucleic acid sequences known in the art and therespective sequences of the cyclin SDS like proteins disclosed hereinare low. Concerning the phenotypic effect in plants, the plants known inthe art, having a mutation in a cyclin SDS protein, have a male sterilephenotype, whereas the plants disclosed herein, having a mutation in acyclin SDS like protein, are male fertile.

A first embodiment of the present invention concerns plant cells, plantparts and plants, characterized in that the plant cells or plants have adecreased activity of a cyclin SDS like protein compared to the plantcells and plants comprising a functional wild type cyclin SDS likeprotein.

In context of the present invention, a “cyclin SDS like protein” shallbe understood to mean a protein which, when its activity is decreased orits expression is entirely knocked-out in a plant, leads (e.g. in aplant homozygous for a mutant nucleotide sequence encoding a cyclin SDSlike protein) to male fertile pollen produced by that plant but at thesame time leads to the production of seedless fruits of said plant, whenself-pollinated.

In context of the present invention, “decreased activity” of a proteinshall mean a decrease in activity of a cyclin SDS like protein whencompared to a corresponding wild type plant cell or a corresponding wildtype plant. Decrease shall in one aspect comprise an entire knock-out ofgene expression, or the production of a loss of function or of adecreased function cyclin SDS like protein, e.g. a truncated SDS likeprotein may have lost function or decreased function. A decrease inactivity can be a decrease in the expression of a gene encoding a cyclinSDS like protein (also referred to as knock-down), or a knock-out of theexpression of a gene encoding a cyclin SDS like protein and/or adecrease in the quantity of a cyclin SDS like protein in the cells or adecrease of function or loss of function in the enzymatic activity of acyclin SDS like proteins in the cells.

“Knock-out” or “entire knock-out” shall be understood that expression ofthe respective gene is not detectable anymore.

“Loss of function (in the enzymatic activity)” shall mean in context ofthe present invention that the protein, although present in amountsequal or similar to a corresponding wild type protein, does not evokeits effect anymore, i.e. mutant alleles when present in homozygous formin a diploid plant, the plant is male fertile but produces only seedlessfruits upon pollination. The terms “non-functional” and “lost activity”shall have the same meaning as “loss of function”. All three terms areused herein interchangeably. Thus, when referring to a cyclin SDS likegene encoding a non-functional protein, the gene may be expressed, butthe encoded protein is not functional, e.g. due to the protein beingtruncated or comprising one or more amino acid replacements, insertionsor deletions compared to the wild SDS like protein.

“Decrease of function (in the enzymatic activity)” or “reduced function”shall mean in context of the present invention that the protein althoughpresent in amounts equal or similar to a corresponding wild typeprotein, does not evoke its effect anymore, i.e. when present inhomozygous form in a diploid plant, the plant is male fertile butproduces only seedless fruits upon pollination.

“Conserved domain” refer to conserved protein domains, such as theCyclin_N (pfam00134) and Cyclin_C domains (pfam02984). These domains cane.g. be found in the Conserved Domain Database of the NCBI (world wideweb at ncbi.nlm.nih.gov/cdd).

“M1 generation” or “M1 plants” in context with the present inventionshall refer to the first generation that is produced directly from themutagenic treatment. A plant grown from seeds treated with a mutagene.g. is a representative of an M1 generation.

“M2 generation” or “M2 plant” shall refer herein to the generationobtained from self-pollination of the M1 generation. A plant grown fromseeds obtained from a self-pollinated M1 plant represents a M2 plant.

The decrease in the expression can, for example, be determined bymeasuring the quantity of RNA transcripts encoding cyclin SDS likeproteins, e.g. using Northern blot analysis or RT-PCR. Here, a reductionpreferably means a reduction in the amount of transcripts by at least50%, in particular by at least 70%, preferably by at least 85% andparticularly preferably by at least 95%.

The decrease in the amount of a cyclin SDS like protein, which resultsin a reduced activity of these proteins in the plant cells or plantsconcerned, can, for example, be determined by immunological methods suchas Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) orRIA (Radio Immune Assay). Here, a decrease preferably means a reductionin the amount of cyclin SDS like proteins by at least 50%, in particularby at least 70%, preferably by at least 85% and particularly preferablyby at least 95%.

Methods for the manufacture of antibodies that react specifically with adesignated protein, i.e. that bind specifically to the said protein, areknown to the person skilled in the art (see, for example, Lottspeich andZorbas (Eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg,Berlin, ISBN 3-8274-0041-4). The manufacture of such antibodies isoffered as a contractual service by several firms.

Concerning the present invention, a decrease of activity of a cyclin SDSlike protein in a plant according to the invention can also bedetermined by the plant phenotype. Plants homozygous for a mutant alleleencoding the cyclin SDS like protein or having a decreased activity of acyclin SDS like protein, produce seedless fruits and are male fertile(produce viable pollen).

In one embodiment the decreased activity of a protein having a cyclinSDS like function is decreased in the plant cells or plants according tothe invention compared to corresponding wild type plant cells or wildtype plants.

In context with the present invention, the term “wild type plant cell”or “wild type plant” means that the plant cells or plants concerned wereused as starting material for the production of the plant cells orplants according to the invention, i.e. their genetic information, apartfrom the introduced (genetic) modification(s) or mutation(s),corresponds to that of a plant cell or plant according to the invention.In one aspect the wild type plant or wild type plant cell is a plantcomprising a fully functional cyclin SDS like protein, e.g. regardingwatermelon plants or plant cells a diploid watermelon plant producingthe protein of SEQ ID NO 2 and producing seeded fruits uponself-pollination. Or regarding melon plants or cells a diploid melonplant producing the protein of SEQ ID NO 6, or regarding cucumber plantsor cells a diploid cucumber plant producing the protein of SEQ ID NO 12,or regarding tomato plants or cells a diploid tomato plant producing theprotein of SE ID NO 19, or regarding pepper plants or cells a diploidplant producing the protein of SEQ ID NO: 20.

In conjunction with the present invention, the term “corresponding”means that, in the comparison of several objects, the objects concernedthat are compared with one another have been kept under the sameconditions. In conjunction with the present invention, the term“corresponding” in conjunction with wild type plant cell or wild typeplant means that the plant cells or plants, which are compared with oneanother, have been raised under the same cultivation conditions, thatthey have the same (cultivation) age and that their genetic information,apart from the introduced (genetic) modification(s) or mutation(s),corresponds to that of a plant cell or plant according to the invention.In case nucleic acid sequences of RNA and DNA molecules are comparedwith each other or said to correspond to each other, it is wellunderstood in the art that a thymine (T) in a DNA molecule is equivalentwith uridine (U) in an RNA molecule. Thus, a T in a DNA sequence is tobe understood to be replaced by a U in an RNA sequence and vice versa,when such molecules are compared with each other.

Preferably in the embodiments according to the invention, the cyclin SDSlike protein of a wild type plant cell, plant part or wild type plant isencoded by nucleic acid molecules selected from the group consisting of:

-   a) nucleic acid molecules, which encode a protein with the amino    acid sequence given under SEQ ID NO 2 (watermelon cyclin SDS like    protein) or SEQ ID NO 6 (melon cyclin SDS like protein) or SEQ ID NO    12 (cucumber cyclin SDS like protein) or SEQ ID NO: 19 (Solanum    lycopersicum cyclin SDS like protein) or SEQ ID NO: 20 (Capsicum    annuum cyclin SDS like protein);-   b) nucleic acid molecules, which encode a protein, the sequence of    which has an identity of at least 58% or at least 60%, preferably at    least 70%, more preferably at least 80%, even further preferred at    least 90%, or particularly preferred at least 95% with the amino    acid sequence given under SEQ ID NO 2 or SEQ ID NO 6 or SEQ ID NO 12    or SEQ ID NO: 19 or SEQ ID NO: 20;-   c) nucleic acid molecules, which comprise the nucleotide sequence    shown under SEQ ID NO 1 or SEQ ID NO 5 or SEQ ID NO 17 or a    complimentary sequence thereof;-   d) nucleic acid molecules, which have an identity of at least 58% or    at least 60% preferably at least 70%, more preferably at least 80%,    even further preferred at least 90% or particularly preferred at    least 95% with the nucleotide sequences described under c);-   e) nucleic acid molecules, which hybridise with at least one strand    of the nucleic acid molecules described under a), b), c), or d)    under stringent conditions;-   f) nucleic acid molecules, the nucleotide sequence of which deviates    from the sequence of the nucleic acid molecules identified under a),    b), c) or d) due to the degeneration of the genetic code; and-   g) nucleic acid molecules, which represent fragments, allelic    variants and/or derivatives of the nucleic acid molecules identified    under a), b), c) or d).

The genomic nucleotide sequence shown under SEQ ID NO 1, and the codingsequence as indicated in SEQ ID NO 1, encodes a wild type cyclin SDSlike protein of Citrullus lanatus (watermelon) having the amino acidsequence as shown under SEQ ID NO 2. SEQ ID NO 5 shows the codingsequence encoding a wild type cyclin SDS like protein from Cucumis melo(melon) having the amino acid sequence as shown under SEQ ID NO 6. SEQID NO 12 shows the wild type cyclin SDS like protein from Cucumissativus (cucumber). SEQ ID NO: 19 shows the wild type cyclin SDS likeprotein of Solanum lycopsersicum (tomato). SEQ ID NO: 20 shows the wildtype cyclin SDS like protein of Capsicum annuum (pepper).

The plant cells, plant parts or plants according to the invention can beplant cells from any species or plants of any species. The plant cellsaccording to the invention can be monocotyledonous and dicotyledonousplant cells, the plants according to the invention can bemonocotyledonous and dicotyledonous plants. Preferably the plant cellsaccording to the invention are plant cells of vegetables (vegetableplant cells) or the plants are vegetable plants, in particularvegetables like tomato, onion, leek, garlic, carrots, pepper, asparagus,artichoke, celeriac, cucumber, melon, gourd, squash, lettuce,watermelon, spinach, cabbage (Brassica oleracea), corn salad, aubergineand okra. More preferred are plant cells from vegetables (vegetableplant cells) or vegetable plants from the Cucurbitaceae family orSolanaceae family. Most preferred plant cells and plants according tothe invention comprise squash (Cucurbita pepo, Cucurbtita maxima,Cucurbita moschata, Lagenaria siceraria), melon (Cucumis melo), cucumber(Cucumis sativus), watermelon (Citrullus lanatus), tomato (Solanumlycopersicum) or pepper (Capsicum annuum) plant cells or plants, inparticular preferred are plant cells from watermelon (Citrullus lanatus)or melon (Cucumis melo) or watermelon (Citrullus lanatus) or melon(Cucumis melo) plants. In one embodiment the plants and plant cells arecultivated plants of these species, such as inbred lines or varietieshaving good agronomic characteristics, especially producing marketableproduce (e.g. fruits) of good quality and uniformity.

Another embodiment of the present invention concerns plants and plantparts comprising plant cells according to the invention.

A further embodiment of the present invention is a plant cell or plant,characterised in that the plant cell or plant has a decreased activityof a cyclin SDS like protein compared to a corresponding wild type plantcell or wild type plant, wherein the cyclin SDS like protein of acorresponding wild type plant cell or wild type plant is encoded bynucleic acid molecules selected from the group consisting of

-   -   a) Nucleic acid molecules, which encode a protein with the amino        acid sequence given under SEQ ID NO 2 (watermelon cyclin SDS        like protein) or SEQ ID NO 6 (melon cyclin SDS like protein) or        SEQ ID NO 12 (cucumber cyclin SDS like protein) or SEQ ID NO: 19        (Solanum lycopersicum cyclin SDS like protein) or SEQ ID NO: 20        (Capsicum annuum cyclin SDS like protein);    -   b) Nucleic acid molecules, which encode a protein, the sequence        of which has an identity of at least 58% or at least 60%,        preferably at least 70%, more preferably at least 80%, even        further preferred at least 90% or particularly preferred at        least 95% with the amino acid sequence given under SEQ ID NO 2        or SEQ ID NO 6 or SEQ ID NO 12 or SEQ ID NO 19 or SEQ ID NO 20;    -   c) Nucleic acid molecules, which comprise the nucleotide        sequence shown under SEQ ID NO 1 or SEQ ID NO 5 or SEQ ID NO 17        or a complimentary sequence thereof;    -   d) Nucleic acid molecules, which have an identity of at least        58% or at least 60%, preferably at least 70%, more preferably at        least 80%, even further preferred at least 90% or particularly        preferred at least 95% with the nucleotide sequences described        under c);    -   e) Nucleic acid molecules, which hybridise with at least one        strand of the nucleic acid molecules described under a), b), c),        or d) under stringent conditions;    -   f) Nucleic acid molecules, the nucleotide sequence of which        deviates from the sequence of the nucleic acid molecules        identified under a), b), c) or d) due to the degeneration of the        genetic code; and    -   g) Nucleic acid molecules, which represent fragments, allelic        variants and/or derivatives of the nucleic acid molecules        identified under a), b), c) or d).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “substantial identity” when they areoptimally aligned by for example the programs GAP or BESTFIT or theEmboss program “Needle” (using default parameters, see below) share atleast a certain minimal percentage of sequence identity (as definedfurther below). These programs use the Needleman and Wunsch globalalignment algorithm for aligning two sequences, over their entirelength, maximizing the number of matches and minimizing the number ofgaps. Generally, the default parameters are used, with a gap creationpenalty=10 and gap extension penalty=0.5 (both for nucleotide andprotein alignments). For nucleotides the default scoring matrix used isDNAFULL and for proteins the default scoring matrix is Blosum62(Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignmentsand scores for percentage sequence identity may for example bedetermined using computer programs, such as EMBOSS, accessible at worldwide web under ebi.ac.uk/Tools/emboss/. Alternatively sequencesimilarity or identity may be determined by searching against databases(e.g. EMBL, GenBank) by using commonly known algorithms and outputformats such as FASTA, BLAST, etc., but hits should be retrieved andaligned pairwise to compare sequence identity. Two proteins or twoprotein domains, or two nucleic acid sequences have “substantialsequence identity” if the percentage sequence identity is at least 58%,60%, 70%, 80%, 90%, 95%, 98%, 99% or more (as determined by Emboss“needle” using default parameters, i.e. gap creation penalty=10, gapextension penalty=0.5, using scoring matrix DNAFULL for nucleic acids anBlosum62 for proteins). Such sequences are also referred to as“variants” or “allelic variants” or “derivatives” herein. Other allelicvariants of the cyclin SDS like protein encoding genes/alleles andcyclin SDS like proteins than the specific nucleic acid and proteinsequences disclosed herein can be identified. So for example cyclin SDSlike proteins having substantial sequence identity to the protein of SEQID NO: 2, or to the protein of SEQ ID NO: 6, or to the protein of SEQ IDNO: 12, or to the protein of SEQ ID NO: 18, or to the protein of SEQ IDNO: 19 are variants of the provided protein.

Allelic variants may exist in other cultivated vegetable plant cells orplants, in particular vegetables like tomato, onion, leek, garlic,carrots, pepper, asparagus, artichoke, gourd, squash, celeriac,cucumber, melon, lettuce, watermelon, spinach, cabbage (Brassica)species, corn salad and okra. Mutations in such allelic variants of thecyclin SDS like protein encoding gene have the same effect on male andfemale fertility and seedless fruit production in other vegetableplants. Particularly allelic variants of a cyclin SDS like gene mayexist in plant cells or plants from the Cucurbitaceae family, like melon(Cucumis melo), cucumber (Cucumis sativus), watermelon (Citrulluslanatus), squash (Cucurbita pepo, Cucurbfita maxima, Cucurbita moschata,Lagenaria siceraria), particular preferred allelic variants of a cyclinSDS like gene may exist in plant cells of watermelon (Citrulluslanatus), melon (Cucumis melo) or cucumber (Cucumis sativus) orwatermelon (Citrullus lanatus) or melon (Cucumis melo) or cucumber(Cucumis sativus) plants. Additionally allelic variants of a cyclin SDSlike gene may also exist in plant cells or plants from the Solanaceaefamily, like tomato (Solanum lycopersicum) or wild relatives of tomato(S. pimpinelli, S. cheesmaniae, S. galapagense, S. pimpinellifolium, S.chmielewskii, S. habrochaites, S. neorickii, and S. pennelli, S.arcanum, S. chilense, S. corneliomulleri, S. huaylasense, and S.peruvianum), pepper (Capsicum annuum), Solanum melongena (aubergine),Solanum tuberosum (potato), etc.

Allelic variants may also exist in other cultivated crop plants, such asfiled crops (e.g. Brassica species, maize, rice, soybean, wheat, barley,cotton, tobacco, coffee, etc.) or fruit crops (e.g. grape, apple, plum,citrus fruits, strawberry, etc.).

It is noted that the cyclin SDS like proteins of the Cucurbitaceae havea high sequence identity to each other (at least 70% for the providedsequences) and the cyclin SDS like proteins of the Solanaceae also havea high sequence identity to each other. On the other hand, the sequenceidentity between Cucurbitaceae and Solanaceae sequences it not high (40%or less), see Table A below.

TABLE A cyclin SDS like protein sequence identity (pairwise alignmentusing Needleman and Wunsch) Watermelon Melon (SEQ ID Cucumber (SEQTomato (SEQ Pepper (SEQ ID (SEQ ID NO 2) NO 6) ID NO 12) ID NO 19) NO20) Watermelon 100%  73% 70% 34% 32% (SEQ ID NO 2) Melon (SEQ ID 100%86% 40% 38% NO 6) Cucumber (SEQ 100%  40% 37% ID NO 12) Tomato (SEQ100%  81% ID NO 19) Pepper (SEQ ID 100%  NO 20)

“Stringent hybridisation conditions” can be used to identify nucleotidesequences, which are substantially identical to a given nucleotidesequence. Stringent conditions are sequence dependent and will bedifferent under different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequences at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridise to a perfectly matched probe. Typicallystringent conditions will be chosen in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least 60° C. Loweringthe salt concentration and/or increasing the temperature increasesstringency. Stringent conditions for RNA-DNA hybridisations (Northernblots using a probe of e.g. 100 nt) are for example those which includeat least one wash in 0.2×SSC at 63° C. for 20 min, or equivalentconditions. Stringent conditions for DNA-DNA hybridisation (Southernblots using a probe of e.g. 100 nt) are for example those which includeat least one wash (usually two) in 0.2×SSC at a temperature of at least50° C., usually about 55° C., for 20 min, or equivalent conditions. Seealso Sambrook et al. (1989) and Sambrook and Russell (2001).

A decrease in the activity of a cyclin SDS like protein in plant cellsor plants according to the invention can also be achieved by a genesilencing effect.

In a further embodiment of the invention the decreased activity of acyclin SDS like protein in plant cells or plants according to theinvention is caused by a gene silencing effect.

The plant cells according to the invention and plants according to theinvention having a decreased activity of a cyclin SDS like protein canbe produced by different methods causing a gene silencing effect knownto the person skilled in the art. These include, for example, theexpression of a corresponding antisense RNA or of a double-stranded RNAconstruct (RNAi technology), the provision of nucleic acid molecules orvectors, which impart a co-suppression effect, the expression of acorrespondingly constructed ribozyme that splits specific transcripts,which code a cyclin SDS like protein.

The decrease of the cyclin SDS like protein activity in plant cells andplants according to the invention can be brought about by expressing anantisense sequence in respective plant cells or plants.

The decrease of the cyclin SDS like protein activity in plant cells andplants according to the invention can be brought about by thesimultaneous expression of sense and antisense RNA molecules (RNAitechnology) of the respective target gene to be repressed, preferably ofthe cyclin SDS like protein encoding gene or allele.

In addition to this, it is known that in planta the formation ofdouble-stranded RNA molecules of promoter sequences can lead in trans tomethylation and transcriptional inactivation of homologous copies ofthis promoter (Mette et al., EMBO J. 19, (2000), 5194-5201). Thedecrease of the cyclin SDS like protein activity in plant cells andplants according to the invention can be brought about by thesimultaneous expression of sense and antisense RNA molecules (RNAitechnology) of promoter sequences initiating transcription of therespective target gene to be repressed, preferably of the cyclin SDSlike protein encoding gene or allele.

Ribozymes have also been described in the art to decrease the expressionof proteins by cleaving RNA molecules encoding the target gene.

Additional discussion of the respective gene silencing technologiesknown to a person skilled in the art will be provided herein furtherbelow and are applicable to the plant cells or plants according to theinvention accordingly.

“Gene silencing effect” refers to a down-regulation or completeinhibition of gene expression of the target gene or gene family.Silenced plant cells or plants produce lower amounts of translationeligible transcripts (including mRNA) for a respective target gene orallele, compared to corresponding wild type plant cells or correspondingwild type plants. The lower amounts of translation eligible transcripts(including mRNA) may be due to targeted degradation of respectivetranscripts.

A “target gene or allele” shall be understood to be the gene or alleleor gene family (or one or more specific alleles of the gene) which hasto be modulated for conferring an organism (e.g. plant cell or plant) toproduce a desired phenotype. Concerning male sterile seedless fruitproducing plants e.g. (a) target gene(s) or (a) target allele(s) is/arethe cyclin SDS line protein encoding gene(s).

In a further embodiment of the invention the decreased activity of acyclin SDS like protein in plant cells or plants according to theinvention is caused by immunomodulation methods.

A further possible way in which to reduce the enzymatic activity ofproteins in plant cells or plants is the so-called immunomodulationmethod. It is known that an in planta expression of antibodies, whichspecifically recognize a plant protein, results in a decrease of theactivity of the proteins concerned. Additional discussion of therespective technology known to a person skilled in the art will beprovided herein further below.

A further embodiment of the invention, are plant cells or plantscharacterised in that they comprise a mutant allele of a cyclin SDS likeprotein encoding gene. The mutant allele of a cyclin SDS like proteinencoding gene can be present in homozygous or heterozygous state. In oneaspect, the mutant allele encodes a cyclin SDS like protein havingdecreased function or loss of function of the encoded mutant protein.The mutant allele may encode a protein with one or more amino acidsreplaced, inserted or deleted, resulting in a protein having decreasedfunction or loss of function compared to the wild type (functional)protein. In one aspect the mutant allele results in a truncated cyclinSDS like protein being produced, which truncated protein has decreasedfunction or loss of function. In another aspect the mutant alleleencodes a protein with one or more amino acids replaced, inserted ordeleted in a conserved domain of the cyclin SDS like protein, such asthe Cyclin_N domain (pfam00134) or the Cyclin_C domain (pfam02984). TheCyclin_N domain and Cyclin_C domain of a protein can be identified bythe skilled person e.g. by a protein against protein BLAST on thewebsite of the NCBI (world wide web at blast.ncbi.nlm.nih.gov) or bysearching in the Conserved Domain Database of the NCBI (world wide webat ncbi.nlm.nih.gov/cdd).

In SEQ ID NO: 2 the Cyclin_N domain ranges from amino acid 388 to aminoacid 463 and the Cyclin_C domain ranges from amino acid 466 to 531.

A protein to protein BLAST provides the Cyclin_N and Cyclin_C domains ofthe search query used, including the alignment of the queried sequencewith the domain. It is noted that “from” a certain number “to” anothernumber includes the end points, i.e. includes the first and last numbermentioned.

So for any of the protein sequences provided herein, or for othervariant sequences (e.g. proteins comprising at least 70%, 80%, 90%, 95%or more sequence identity to any of the protein sequences providedherein, e.g. SEQ ID NO: 2, 6, 12, 19 or 20) the Cyclin_N and Cyclin_Cdomains can be determined.

In SEQ ID NO 6 the Cyclin_N domain ranges from amino acid 351 to aminoacid 481 and the Cyclin_C domain ranges from amino acid 486 to aminoacid 577.

In SEQ ID NO: 12 the Cyclin_N domain ranges from amino acid 343 to aminoacid 473 and the Cyclin_C domain ranges from amino acid 478 to aminoacid 569.

In SEQ ID NO: 19 the Cyclin_N domain ranges from amino acid 362 to aminoacid 494 and the Cyclin_C domain ranges from amino acid 499 to 584.

In SEQ ID NO: 20 the Cyclin_N domain ranges from amino acid 332 to aminoacid 464 and the Cyclin_C domain ranges from amino acid 469 to 554.

Plants in which the mutant allele of a cyclin SDS like protein encodinggene is present in a heterozygous state, will produce seeds and are malefertile. Thus, those plants can be used to introduce the mutant alleleof a cyclin SDS like protein encoding gene into other plants or they canbe used to introduce further traits into the plant in which the mutantallele of a cyclin SDS like protein encoding gene is present. Theseplants can also be used to propagate plants comprising a mutant alleleof a cyclin SDS like encoding gene. 50% of the self-pollinated offspringin each case will still carry the mutant allele of a cyclin SDS likeprotein encoding gene in a heterozygous state. Therefore, plants inwhich the mutant allele of a cyclin SDS like protein encoding gene ispresent are useful e.g. in breeding.

Therefore, one embodiment of the invention concerns plant cells orplants according to the invention which are heterozygous for a mutantallele of a cyclin SDS like protein encoding gene.

In a preferred embodiment of the invention, the decreased activity of acyclin SDS like protein in plant cells or plants according to theinvention is due to or caused by or is the effect of a mutant allele ofa cyclin SDS like protein encoding gene being present in plant cells orplants, respectively.

In one aspect, the plant cells or plants according to the invention arehomozygous for the mutant allele of a cyclin SDS like protein encodinggene encoding a decrease of function of a cyclin SDS like protein or aloss of function of a cyclin SDS like protein. Plants according to theinvention being homozygous for a mutant allele of a cyclin SDS likeprotein encoding gene produce seedless fruits upon pollination with ownpollen or pollen obtained from a different plant (e.g. from a wild typeplant).

Another embodiment of the invention therefore concerns plant cells orplants according to the invention which are homozygous for a mutantallele of a cyclin SDS like protein encoding gene.

A mutant allele of a cyclin SDS like protein encoding gene causes aplant to be male fertile but producing seedless fruits, when the plantis homozygous for the mutant allele. Concerning the embodiments of theinvention, the mutation in the mutant allele of a cyclin SDS likeprotein encoding gene can be any mutation, including deletions,truncations, insertions, point mutations, nonsense mutations, missenseor non-synonymous mutations, splice-site mutations, frame shiftmutations and/or mutations in regulatory sequences. Preferably themutation in the mutant allele of a cyclin SDS like protein encoding geneis a point mutation and/or splice-site mutation. The mutation can occurin a DNA sequence comprising the coding sequence of a cyclin SDS likeprotein encoding gene or in a RNA sequence encoding a cyclin SDS likeprotein or it can occur in the amino acid of a cyclin SDS like protein.Concerning a DNA sequence of a cyclin SDS like protein encoding gene themutation can occur in the coding sequence (cds, composed of the exons)or it can occur in non-coding sequences like 5′- and 3′-untranslatedregions, introns, promoters, enhancers etc. of a cyclin SDS like proteinencoding gene. In respect to RNA encoding a cyclin SDS like protein themutation can occur in the pre-mRNA or the mRNA. In one aspect the mutantallele results in the protein having a loss of function or decrease offunction due to one or more amino acids being replaced, inserted and/ordeleted, for example resulting in one or more amino acids beingreplaced, inserted or deleted in the conserved Cyclin_N and/or Cyclin_Cdomain. For example, truncation of the protein to cause deletion of theCyclin_C domain, or part thereof, or the Cyclin_N domain and theCyclin_C domain or part of the Cyclin_N domain and the Cyclin_C domainwill result in a loss of function or decrease of function of theprotein.

A further embodiment of the invention therefore concerns plant cells orplants according to the invention comprising a mutant allele of a cyclinSDS like protein encoding gene characterized in that the mutant allelecomprises or effects one or more of the mutations selected from thegroup consisting of

-   a) a deletion, truncation, insertion, point mutation, nonsense    mutation, missense or non-synonymous mutation, splice-site mutation,    frame shift mutation in the genomic sequence;-   b) a mutation in one or more regulatory sequences;-   c) a deletion, truncation, insertion, point mutation, nonsense    mutation, missense or non-synonymous mutation, splice-site mutation,    frame shift mutation in the coding sequence;-   d) a deletion, truncation, insertion, point mutation, nonsense    mutation, missense or non-synonymous mutation, splice-site mutation,    frame shift mutation in the pre-mRNA or mRNA; and/or-   e) a deletion, truncation, insertion or replacement of one or more    amino acids in the cyclin SDS like protein.

Compared to SEQ ID NO 1, one of the mutant alleles of a cyclin SDS likeprotein encoding gene disclosed herein as one embodiment (present in theEMB1 mutant watermelon plant), has a point mutation (replacement of G byA) at nucleotide position 2185 in SEQ ID NO 1. mRNA transcribed from thedisclosed mutant allele of a cyclin SDS like protein encoding gene isshown under SEQ ID NO 3. Corresponding nucleotides at position 2186 to2201 of the wild type allele of the cyclin SDS like protein encodinggene shown under SEQ ID NO 1 are not present in the mRNA shown under SEQID NO 3. Thus, the point mutation found in the mutant allele of thecyclin SDS like protein encoding gene causes a deletion of 16nucleotides in mRNA transcribed from the mutant allele compared to themRNA transcribed from the corresponding wild type allele. The deletionin mRNA transcribed from the mutant allele is explained by mutation of asplice site resulting in alternative splicing of the respective mRNA. Inaddition, the deletion of 16 nucleotides in the mRNA transcribed fromthe disclosed mutant allele of the cyclin SDS like protein encoding genecauses a frame shift in the reading frame of the mRNA transcribed fromthe mutant allele compared to mRNA transcribed from the correspondingwild type allele. The protein translated from the wild type allele ofthe cyclin SDS like protein encoding gene is shown under SEQ ID NO 2.The protein translated from the mutant allele of the cyclin SDS likeprotein encoding gene is shown under SEQ ID NO 4. The correspondingnucleotide sequence encoding amino acids 358 to 363(Ile-Leu-Arg-Phe-Glu-Glu) in SEQ ID NO 2 is not present in SEQ ID NO 4and due to the frame shift in the reading frame the rest of the aminoacid sequence is different and amino acids 364 to 562 in SEQ ID NO 2 arereplaced by the 8 aberrant amino acids Asn-Trp-Thr-Met-Lys-Lys-Pro-Ilein SEQ ID NO 4 (i.e. amino acids 358 to 365 of SEQ ID NO 4). The mutantcyclin SDS like protein is much shorter, only 365 amino acids, comparedto the wild type protein of 562 amino acids. Thus, the amino acidsequence of the mutant cyclin SDS like protein as shown under SEQ ID NO4 comprises amino acid deletions and amino acid replacements comparedthe wild type cyclin SDS like protein shown under SEQ ID NO 2.Furthermore, the frame shift in the reading frame of the mRNAtranscribed from the mutant allele of a SDS like protein encoding genecauses a non-sense mutation creating a pre-mature stop codon(nucleotides 1096 to 1098 in SEQ ID NO 3), leading to the amino acidsequence encoded by the mutant allele being truncated by 197 amino acidsat the C-terminus when compared to the corresponding amino acid sequenceencoded by the wild type allele shown under SEQ ID NO 2. Thus, comparedto the wild type protein, the 205 amino acids of the wild typeC-terminus are replaced by 8 different (aberrant) amino acids at theC-terminal end due to the frame shift, resulting in a mutant proteinthat is 197 amino acids shorter than the wild type protein. The mutantprotein is, therefore, truncated compared to the wild type protein, as205 amino acids of the wild type C-terminus are missing in the mutantprotein. Of the wild type protein only the amino acids of exon 1 (aminoacids 1 to 357 of SEQ ID NO: 2) are still present in the mutant protein.This means that also the conserved protein domains Cyclin_N and Cyclin_Care not present, meaning that the mutant protein has no function (i.e.is the mutant allele is a loss of function allele) or a decrease offunction.

In summary, specifically disclosed herein for exemplifying theapplication is in one aspect a nucleic acid sequence of a mutant alleleof a cyclin SDS like protein encoding gene which has a point mutation(nucleotide replacement) compared to the nucleic acid sequence of thecorresponding wild type cyclin SDS like protein encoding gene. The pointmutation in the mutant allele of the cyclin SDS like protein encodinggene causes a splice-site mutation leading to alternative splicing ofthe respective pre-mRNA. The alternative splicing causes a frame shiftin the open reading frame of the mRNA transcribed from the mutant alleleof the cyclin SDS like protein encoding gene. The frame shift in theopen reading frame of the mRNA transcribed from the mutant allele of thecyclin SDS like protein encoding gene causes deletion of nucleotides,replacement of nucleotides (missense or non-synonymous mutations) andthe creation of a non-sense mutation creating a pre-mature stop codon inthe mRNA transcribed from the mutant allele of the cyclin SDS likeprotein encoding gene compared to the mRNA transcribed from thecorresponding wild type cyclin SDS like protein encoding gene. Therespective amino acid sequence of the protein translated from the mRNAtranscribed from the mutant cyclin SDS like protein encoding gene showsdeletion of amino acids, replacement of amino acids and truncation ofthe amino acid sequence at the C-terminus compared to the amino acidsequence translated from the mRNA transcribed from the correspondingwild type cyclin SDS like protein encoding gene. As the point mutationis in the first intron, the amino acids encoded by exon 2, exon 3 andexon 4 of the wild type cyclin SDS like protein are missing in themutant, i.e. only the amino acids encoded by exon 1 of the wild type SDSlike protein are present in the mutant protein.

In watermelon exon 1 of the cyclin SDS like protein encodes amino acids1 to 357 of SEQ ID NO 2; in melon exon 1 of the cyclin SDS like proteinencodes amino acids 1 to 338 in SEQ ID NO: 6; in cucumber exon 1 of thecyclin SDS like protein encodes amino acids 1 to 330 in SEQ ID NO: 12;in tomato exon 1 of the cyclin SDS like protein encodes amino acids 1 to350 in SEQ ID NO 19; in pepper exon 1 of the cyclin SDS like proteinencodes amino acids 1 to 320 in SEQ ID NO 20.

In addition to the watermelon EMB1 mutant plant described herein above(and in the examples), another watermelon plant, comprising a differentmutation in the cyclin SDS like protein encoding gene, has beengenerated by mutagenesis. The mutant comprises a C (Cytosine) to T(Thymine) nucleotide substitution at nucleotide 1687 of SEQ ID NO: 1,leading to the codon ‘cag’ (encoding the amino acid glutamine, aminoacid 224 of the wild type protein) being changed into ‘tag’, which is astop codon. The mutant cDNA is shown in SEQ ID NO 17 and the truncatedprotein, comprising only part of the amino acids encoded by exon 1 (i.e.only amino acids 1 to 223 instead of amino acids 1 to 357), is shown inSEQ ID NO 18. Like in the EMB1 mutant plant, the two conserved domains,the Cyclin_N and Cyclin_C domains, are missing in the mutant protein.Also this mutant results in a loss of function of the cyclin SDS likeprotein (or at least in a decreased of function).

In one aspect of the invention the mutant allele of a cyclin SDS likeprotein encoding gene has a mutation leading to one or more or all ofthe amino acids encoding the Cyclin_N and/or Cyclin_C domain beingdeleted or being replaced by different amino acids than in the wildtype. In one aspect the mutant allele results in a truncated proteinlacking all or part of the Cyclin_N and/or all or part of the Cyclin_Cdomain. For example, the mutant allele contains a mutation leading to apremature stop codon, whereby all or part of the Cyclin_N and/or all orpart of the Cyclin_C domain are not present anymore in the resultingprotein.

In one aspect the mutant allele is a mutant allele of the watermeloncyclin SDS like gene of SEQ ID NO: 1 and leads to the protein of SEQ IDNO: 4 or to the protein of SEQ ID NO: 18.

In one aspect of the invention the mutant allele of a cyclin SDS likeprotein encoding gene has a mutation leading to the amino acids encodingexons 2, 3 and/or 4 of the wild type protein being absent. Thus in oneaspect the mutant allele encodes a truncated cyclin SDS like protein/ora protein comprising a deletion, which lacks the amino acids encoded byexons 2, 3 and/or 4 of the wild type protein, i.e. it lacks amino acids358 to 413 of SEQ ID NO 2 (exon 2) or amino acids 339 to 394 of SEQ IDNO: 6 (exon 2), amino acids 331 to 386 of SEQ ID NO 12 (exon 2), aminoacids 351 to 407 of SEQ ID NO 19 (exon 2), amino acids 321 to 377 of SEQID NO: 20 (exon 2), and/or amino acids 414 to 469 of SEQ ID NO 2 (exon3) or amino acids 395 to 493 of SEQ ID NO 6 (exon 3) or amino acids 387to 485 of SEQ ID NO 12, amino acids 408 to 506 of SEQ ID NO 19 (exon 3),amino acids 378 to 476 of SEQ ID NO 20 (exon 3), and/or amino acids 470to 562 of SEQ ID NO 2 (exon 4) or amino acids 494 to 577 of SEQ ID NO: 6(exon 4) or amino acids 486 to 569 of SEQ ID NO 12 (exon 4), amino acids507 to 590 of SEQ ID NO 19 (exon 4), amino acids 477 to 560 of SEQ ID NO20 (exon 4). Optionally the mutant allele encodes a truncated cyclin SDSlike protein/or a protein comprising a deletion, which further lacks allor part of the amino acids encoding exon 1, i.e. amino acids 1 to 357 ofSEQ ID NO 2 or amino acids 1 to 338 in SEQ ID NO: 6 or amino acids 1 to330 in SEQ ID NO 12 or amin acids 1 to 350 of SEQ ID NO 19, or aminoacids 1 to 320 of SEQ ID NO 20. For corresponding amino acid regionsencoded by exons 1, 2, 3 and 4 of other cyclin SDS like proteins, e.g.of orthologs from other species, can be identified by pairwise alignmentof the genomic DNA or the amino acid sequences. It is noted that onlyfor SEQ ID NO 2 the exons were determined to be real exons (separated byintrons on the genomic DNA), while for the other sequences the exonswere determined by alignment and may not be the real exons, but are therather the amino acids corresponding to the exons of SEQ ID NO 2 and cantherefore also be simply referred to as amino acid regions of theprotein.

In a preferred embodiment of the invention the mutant allele of a cyclinSDS like protein encoding gene has, or results in, a mutation at the5′-end of its coding sequence (encoding the N-terminus of the protein).More preferred the mutant allele of a cyclin SDS like protein encodinggene has, or results in, a point mutation and/or a truncation at the5′-end of its coding sequence. It is well known in the art that amutation in the start codon (ATG) of a gene will have the effect thatthe respective gene is not translated into a respective full-lengthprotein. For translation the next possible start codon (ATG) may beused, but this will lead to a truncation of the amino acid sequence ofthe protein at the N-terminus, in case the next ATG appears in the samereading frame or to the production of a protein having a different aminoacid sequence. In both cases, the respective mutation at the 5′-end willlead to the production of a protein having decreased or lost enzymaticactivity. In a preferred embodiment of the invention, the mutant alleleof a cyclin SDS like protein encoding gene has a mutation in the startcodon. The mutation in the start codon can be a point mutation in any ofits three nucleotides or a deletion/truncation of at least the first, atleast the first and the second or at least all three nucleotides of thestart codon.

Further preferred embodiments of the invention are mutant alleles of acyclin SDS like protein encoding gene which results in a deletion at the3′-end of its coding sequence (encoding the C-terminus of the protein).As the conserved Cyclin_C domain is present at the C-terminus of theprotein, a truncation which results in part of the Cyclin_C domain beingabsent (e.g. 1, 2, 3, 4, 5 or more amino acids of the Cyclin_C domain oreven all of the Cyclin_C domain) will result in the protein havingreduced function or no function. A longer truncation at the C-terminus,will even result in part or all of the Cyclin_N domain being deleted,which will likewise result in the protein having reduced or no function.There are only five amino acids between the Cyclin_C and Cyclin_Ndomain. A truncation of about 90 or more amino acids of the C-terminusresults in most of the cyclin SDS like proteins missing the Cyclin_Cdomain and longer truncations of 95, 100, 110 or more amino acids willresult in the Cyclin_N domain being deleted at least partially. Asmentioned before, such truncations are encompassed herein, as leading toa protein of reduced function or no function. As mentioned previously,to test whether the protein has reduced function or no function, themutant plant being homozygous for the mutant allele can be testedphenotypically to see if the expected phenotype occurs.

Preferably the mutant alleles of a cyclin SDS like protein encoding generesults in a truncation of at least 10, 20, 30, 40 or 50 nucleotides,preferably at least 100 nucleotides, more preferred of at least 200nucleotides, even more preferred of at least 300 nucleotides, furtherpreferred of at least 400 nucleotides and most preferred of at least 500nucleotides, in particular preferred of at least 615 nucleotides at the3′end of the protein coding sequence. A truncation by 591 nucleotidesfrom a coding sequence translates into a truncation by 197 amino acidsfor the respective protein sequence. A preferred example for a cyclinSDS like protein having a truncation by 197 amino acids compared to theamino acid sequence of a corresponding wild type cyclin SDS like protein(SEQ ID NO 2) is shown under SEQ ID NO 4.

Another example is a truncation of 339 amino acids (i.e. 1017nucleotides of the coding region) compared to the corresponding wildtype SDS like protein (SEQ ID NO 2) is shown in SEQ ID NO 18.

In one embodiment, the mutant allele of a cyclin SDS like proteinencoding gene has any of the mutations mentioned above in a nucleic acidsequence encoding the protein of SEQ ID NO: 2 (resulting in the encodedprotein comprising a deletion or truncation compared to the wild type),or in any nucleic acid sequence encoding a protein comprising at least70%, 80%, 90%, 95% or more amino acid sequence identity to SEQ ID NO 2,such as for example SEQ ID NO 6, which has 71% sequence identity to SEQID NO 2.

In another embodiment, the mutant allele of a cyclin SDS like proteinencoding gene has any of the mutations mentioned above in a nucleic acidsequence encoding the protein of SEQ ID NO: 6 (resulting in the encodedprotein comprising a deletion or truncation compared to the wild type),or in any nucleic acid sequence encoding a protein comprising at least70%, 80%, 90%, 95% or more amino acid sequence identity to SEQ ID NO 6.

In another embodiment, the mutant allele of a cyclin SDS like proteinencoding gene has any of the mutations mentioned above in a nucleic acidsequence encoding the protein of SEQ ID NO: 12 (resulting in the encodedprotein comprising a deletion or truncation compared to the wild type),or in any nucleic acid sequence encoding a protein comprising at least70%, 80%, 90%, 95% or more amino acid sequence identity to SEQ ID NO:12.

In another embodiment, the mutant allele of a cyclin SDS like proteinencoding gene has any of the mutations mentioned above in a nucleic acidsequence encoding the protein of SEQ ID NO: 19 (resulting in the encodedprotein comprising a deletion or truncation compared to the wild type),or in any nucleic acid sequence encoding a protein comprising at least70%, 80%, 90%, 95% or more amino acid sequence identity to SEQ ID NO:19.

In another embodiment, the mutant allele of a cyclin SDS like proteinencoding gene has any of the mutations mentioned above in a nucleic acidsequence encoding the protein of SEQ ID NO: 20 (resulting in the encodedprotein comprising a deletion or truncation compared to the wild type),or in any nucleic acid sequence encoding a protein comprising at least70%, 80%, 90%, 95% or more amino acid sequence identity to SEQ ID NO:20.

In a further preferred embodiment, the mutant allele of a cyclin SDSlike protein encoding gene has any of the mutations mentioned above inthe nucleic acid sequence shown under SEQ ID NO 1 or in a sequencehaving an identity of at least 58% or 60%, preferably at least 70%, morepreferably at least 80%, even further preferred at least 90% orparticularly preferred at least 95% with the nucleic acid sequence shownunder SEQ ID NO 1. In one aspect a mutant allele of a cyclin SDS likeprotein encoding gene comprises a mutation in the nucleic acid sequenceshown under SEQ ID NO 1 or in a variant sequence having an identity ofat least 58% or 60%, preferably at least 70%, more preferably 80%, evenfurther preferred 90% or particularly preferred 95% with the nucleicacid sequence shown under SEQ ID NO 1 wherein the nucleotide guanine (G)at nucleotide position number 2185 in SEQ ID NO 1 is replaced by adenine(A), cytosine (C) or thymine (T), most preferably the nucleotide guanine(G) at nucleotide position number 2185 in SEQ ID NO 1, or the equivalentnucleotide in a variant sequence, is replaced by adenine (A). Mostpreferably, the mutant allele of a cyclin SDS like protein encoding genehas the nucleotide acid sequence shown under SEQ NO 1 apart from thatthe nucleotide guanine (G) at nucleotide position number 2185 in SEQ IDNO 1 is replaced by adenine (A), cytosine (C) or thymine (T), mostparticularly preferred the nucleotide guanine (G) at nucleotide positionnumber 2185 in SEQ ID NO 1 is replaced by adenine (A). In a differentaspect a mutant allele of a cyclin SDS like protein encoding genecomprises a mutation in the nucleic acid sequence shown under SEQ ID NO1 or in a variant sequence having an identity of at least 58% or 60%,preferably at least 70%, more preferably 80%, even further preferred 90%or particularly preferred 95% with the nucleic acid sequence shown underSEQ ID NO 1 wherein in the mutant allele of a SDS like protein encodinggene the nucleotide cytosine (C) at nucleotide 1687, or the equivalentnucleotide in a variant sequence, is replaced by a different nucleotide,preferably by thymine (T).

A different embodiment of the invention concerns plant cells, plantparts or plants comprising or synthesising an mRNA encoding a cyclin SDSlike protein, wherein the mRNA encoding a cyclin SDS like protein hasone or more mutations selected from the group consisting of

-   a) a deletion mutation-   b) a missense or non-synonymous mutation;-   c) a frame shift mutation; and/or-   d) a non-sense mutation.

In a preferred embodiment of the invention, plant cells, plant parts orplants according to the invention comprise or synthesise an mRNAencoding a cyclin SDS like protein having one or more of the mutationsselected from the group consisting of

-   a) a deletion mutation-   b) a missense or non-synonymous mutation;-   c) a frame shift mutation; and/or-   d) a non-sense mutation.

Concerning deletions and replacements of one or more nucleotides, plantcells or plants according to the invention preferably comprise orsynthesise an mRNA encoding a cyclin SDS like protein, wherein the mRNAcomprises a deletion of at least 1, at least 2, at least 4, at least 5,at least 7, at least 8, at least 10, at least 11, at least 13, at least14 or preferably at least 16 nucleotides compared to mRNA encoding awild type cyclin SDS like protein. In one aspect the nucleotides(s)deleted in the mRNA are one or more nucleotides of exon 1, exon 2, exon3 and/or exon 4 of the cyclin SDS like protein and/or the nucleotide(s)deleted in the mRNA are one or more nucleotides of the Cyclin_N orCyclin_C domain of the cyclin SDS like protein. In one aspect thenucleotides are nucleotides of exon 2, e.g. one or more or allnucleotides starting at nucleotide 2186 and ending at nucleotide 2201 ofSEQ ID NO 1.

Preferably, plant cells or plants according to the invention comprise orsynthesise an mRNA encoding a cyclin SDS like protein, characterized inthat the mRNA comprises a frame shift mutation and/or a non-sensemutation. The non-sense mutation creates a pre-mature stop codon andthus a truncation of the mRNA coding sequence. A preferred embodiment ofthe invention concerns plant cells or plants according to the inventioncomprising an mRNA encoding a cyclin SDS like protein, characterized inthat the mRNA comprises a truncation of the coding sequence. Thetruncation of the mRNA coding sequence of a cyclin SDS like proteinpreferably is a truncation of at least 100 nucleotides, preferred of atleast 200 nucleotides, more preferred of at least 300 nucleotides, evenmore preferred of at least 400 nucleotides and further more preferred ofat least 500 nucleotides, in particular preferred of at least 591nucleotides compared to mRNA encoding a wild type cyclin SDS likeprotein. In one aspect the truncation of the mRNA coding sequenceresults in exon 2, 3 and 4 to be absent; or exon 3 and 4 to be absent;or exon 4 to be absent. In another aspect the truncation of the mRNAcoding sequence results in all or part of exon 1, all of exon 2, all ofexon 3 and all of exon 4 to be absent. In another aspect, theframe-shift mutation results in all or part of exon 2 to be in adifferent reading frame. In a different aspect the frame-shift mutationresults in all or part of exon 3 and/or exon 4 to be in a differentreading frame. The frame shift may be caused by the deletion of one ormore nucleotides (any number which is not a multitude of three, such as1, 2, 4, 5, 7, 8, 10, etc.), whereby the reading frame is changed.

In a preferred embodiment, the plant cells, plant parts or plantsaccording to the invention comprise or synthesise an mRNA encoding acyclin SDS like protein which mRNA has at least 58% or at least 60%,preferably at least 70%, more preferably at least 80%, even furtherpreferred at least 90% or particularly preferred at least 95% sequenceidentity with the corresponding coding sequence indicated in SEQ ID NO 1or SEQ ID NO 5 or SEQ ID NO 17 with the prerequisite that the mRNAencoding a cyclin SDS like protein comprises a non-sense mutation or apre-mature stop codon. In one embodiment the stop codon is in exon 1 ofSEQ ID NO: 1, e.g. at nucleotides 1687 to 1689. In a further preferredembodiment, the plant cells, plant parts or plants according to theinvention comprise or synthesise an mRNA encoding a cyclin SDS likeprotein which mRNA has at least 58% or 60%, preferably at least 70%,more preferably at least 80%, even further preferred at least 90% orparticularly preferred at least 95% sequence identity with the codingsequence indicated in SEQ ID NO 3, with the prerequisite thatnucleotides 1096 to 1098 in SEQ ID NO 3 represent a stop codon. In themost preferred embodiment of the invention, plant cells or plantsaccording to the invention comprise or synthesise an mRNA encoding acyclin SDS like protein which mRNA has the sequence shown under SEQ IDNO 3.

In another embodiment of the invention, plant cells or plants accordingto the invention comprise or synthesise an mRNA encoding a cyclin SDSlike protein having one or more mutations, wherein the mRNA istranscribed from a mutant allele of a cyclin SDS like protein encodinggene. Comprised by these embodiments of the invention are plant cells,plant parts or plants according to the invention comprising orsynthesising an mRNA transcribed from a mutant allele of a cyclin SDSlike protein encoding gene, characterized in that the mRNA comprises adeletion mutation and/or a missense or non-synonymous mutation and/or aframe shift mutation and/or a non-sense mutation, compared to thecorresponding (DNA) coding sequence of the mutant allele of the cyclinSDS like protein encoding gene from which the mRNA is transcribed. Thus,in one aspect any mutation which affects pre-mRNA splicing isencompassed, i.e. which modifies the normal pre-mRNA splicing process,thereby leading to a different mRNA molecule.

Concerning deletion mutations, plant cells or plants according to theinvention in one aspect comprise or synthesise an mRNA transcribed froma mutant allele of a cyclin SDS like protein encoding gene, wherein themRNA comprises a deletion of at least 1, at least 2, at least 4, atleast 5, at least 7, at least 8, at least 10, at least 11, at least 13,at least 14 or at least 16 nucleotides compared to the corresponding(DNA) coding sequence of the mutant allele of the cyclin SDS likeprotein encoding gene from which the mRNA is transcribed. In one aspectthe nucleotides(s) deleted in the mRNA are one or more nucleotides ofexon 1, exon 2, exon 3 and/or exon 4 of the cyclin SDS like protein. Inone aspect the nucleotides are nucleotides of exon 2, e.g. one or moreor all nucleotides starting at nucleotide 2186 and ending at nucleotide2201 of SEQ ID NO 1.

Preferably, plant cells or plants according to the invention comprise orsynthesise an mRNA transcribed from a mutant allele of a cyclin SDS likeprotein encoding gene, characterized in that the mRNA comprises a frameshift mutation and/or a non-sense mutation compared to the (DNA) codingsequence of the mutant allele of the cyclin SD S like protein encodinggene from which the mRNA is transcribed. The non-sense mutation createsa pre-mature stop codon in the mRNA which causes a 3′-end truncation ofthe mRNA coding sequence and a truncation of a cyclin SDS like proteinat the C-terminus. A preferred embodiment of the invention thereforeconcerns plant cells or plants according to the invention comprising orsynthesising an mRNA transcribed from a mutant allele of a cyclin SDSlike protein encoding gene, characterized in that the mRNA comprises atruncation of the coding sequence compared to the (DNA) coding sequenceof the mutant allele of the cyclin SDS like protein encoding gene fromwhich the mRNA is transcribed. The truncation of the mRNA codingsequence of a cyclin SDS like protein encoding gene preferably is atruncation of at least 100 nucleotides, more preferred of at least 200nucleotides, even more preferred of at least 300 nucleotides, morepreferred of at least 400 nucleotides and more preferred of at least 500nucleotides, in particular preferred of at least 591 compared to the(DNA) coding sequence of the mutant allele of the cyclin SDS likeprotein encoding gene from which the mRNA is transcribed. In one aspectthe truncation of the mRNA coding sequence results in exon 2, 3 and 4 tobe absent; or exon 3 and 4 to be absent; or exon 4 to be absent. Inanother aspect the truncation of the mRNA coding sequence results in allor part of exon 1, all of exon 2, all of exon 3 and all of exon 4 to beabsent. In another aspect, the frame-shift mutation results in all orpart of exon 2 to be in a different reading frame. In a different aspectthe frame-shift mutation results in all or part of exon 3 and/or exon 4to be in a different reading frame. The frame shift may be caused by thedeletion of one or more nucleotides (any number which is not a multitudeof three, such as 1, 2, 4, 5, 7, 8, 10, etc.), whereby the reading frameis changed.

In a further preferred embodiment the plant cells or plants according tothe invention comprise or synthesise an mRNA having an identity of atleast 58% or 60% preferably at least 70%, more preferably at least 80%,even further preferred at least 90% or particularly preferred at least95% with the corresponding (DNA) coding sequence indicated in SEQ ID NO1 with the prerequisite that the mRNA sequence comprises at least anon-sense mutation or a pre-mature stop codon compared to thecorresponding (DNA) coding sequence indicated in SEQ ID NO 1,preferably, the non-sense mutation occurs at nucleotide positions 1096to 1098 in SEQ ID NO 3. In one aspect the premature stop codon is atnucleotides 1687 to 1689 of SEQ ID NO: 1. In a particular preferredembodiment of the invention, the plant cells or plants according to theinvention comprise an mRNA having the nucleotide sequence shown underSEQ ID NO 3.

An “mRNA coding sequence” shall have the common meaning herein. An mRNAcoding sequence corresponds to the respective DNA coding sequence of agene/allele apart from that thymine (T) is replaced by uracil (U).

For any of the above described mutations or combinations of mutations(e.g. nucleotide deletion resulting in a frame shift), it is understoodthat they result in causing a decrease of function or a loss of functionin activity of the cyclin SDS like protein in plant cells, plant partsor plants according to the invention.

Another embodiment of the invention concerns plant cells, plant parts orplants, comprising or synthesising a cyclin SDS like protein,characterized in that the amino acid sequence of the cyclin SDS likeprotein comprises a mutation compared to the corresponding wild typecyclin SDS like protein. The mutation in the cyclin SDS like proteincauses a decrease or loss of function in activity of a cyclin SDS likeprotein in plant cells, plant parts or plants according to theinvention.

Particularly preferred are plant cells, plant parts or plants accordingto the invention comprising or synthesising a cyclin SDS like protein,characterized in that the amino acid sequence of the cyclin SDS likeprotein comprises a mutation compared to the corresponding wild typecyclin SDS like protein.

The mutation in the cyclin SDS like protein can be an amino acidreplacement, insertion, deletion and/or truncation compared to the aminoacid sequence of a wild type cyclin SDS like protein. In a preferredembodiment of the invention the amino acid sequence of the cyclin SDSlike protein comprises a deletion or truncation, more preferred atruncation at the N-terminus and/or C-terminus, even more preferred atruncation at the C-terminus. Preferably at least 10, at least 25,preferably at least 50, 60, 70, 80, 90 or 100, more preferably at least150 and even more preferred at least 197 or at least 200, 250, 300 or339 amino acids are missing from the N-terminal end or C-terminal end ofthe amino acid sequence compared to the corresponding wild type cyclinSDS like protein. Concerning the C-terminus, the mutation in the cyclinSDS like protein is a truncation of at least 25, preferably at least 50,60, 70, 80, 90, preferably at least 100, more preferably at least 150and even more preferred of at least 197 or at least 200, 250, 300 or 339amino acids compared to the corresponding wild type cyclin SDS likeprotein. In another aspect the mutation in the cyclin SDS like proteinresults in the amino acids encoded by exon 2, 3 and 4 to be absent; orthe amino acids encoded by exon 3 and 4 to be absent; or in amino acidsencoded by exon 4 to be absent. In another aspect the mutation resultsin all or part of the amino acids encoded by exon 1, all amino acidsencoded by exon 2, all amino acids encoded by exon 3 and all amino acidsencoded by exon 4 to be absent. In another aspect, the mutation resultsin all or part of amino acids encoded by exon 2 to be replaced bydifferent amino acids (e.g. due to a reading frame shift). In adifferent aspect the mutation results in amino acids encoded by all orpart of exon 3 and/or exon 4 to be replaced by different amino acids(e.g. due to a reading frame shift). In a different aspect the mutationresults in all or part of the Cyclin_C and/or Cyclin_N domain beingabsent. In yet a different aspect the mutation results in the Cyclin_Cand/or Cyclin_N domain comprising one or more amino acids replaced,inserted or deleted.

Further provided are plant cells, plant parts or plants according to theinvention comprising or synthesising a cyclin SDS like protein which hasat least 58% or at least 60%, preferably at least 70%, more preferablyat least 80%, even further preferred at least 90% or particularlypreferred at least 95% identity with the amino acid sequence shown underSEQ ID NO 4 or under SEQ ID NO 18. In the most preferred embodiment theprotein comprised or synthesized in the plant cells, plant parts orplants has the amino acid sequence shown under SEQ ID NO 4 or under SEQID NO: 18.

A further embodiment of the invention therefore concerns plant cells orplants selected from the species watermelon, melon, cucumber, tomato andpepper comprising a mutant allele of a cyclin SDS like protein encodinggene characterized in that the mutant allele comprises or effects one ormore of the mutations selected from the group consisting of

-   -   a) a deletion, truncation, insertion, point mutation, nonsense        mutation, missense or non-synonymous mutation, splice-site        mutation, frame shift mutation in the genomic sequence;    -   b) a mutation in one or more regulatory sequences;    -   c) a deletion, truncation, insertion, point mutation, nonsense        mutation, missense or non-synonymous mutation, splice-site        mutation, frame shift mutation in the coding sequence;    -   d) a deletion, truncation, insertion, point mutation, nonsense        mutation, missense or non-synonymous mutation, splice-site        mutation, frame shift mutation in the pre-mRNA or mRNA; and/or    -   e) a deletion, truncation, insertion or replacement of one or        more amino acids in the cyclin SDS like protein.

The above mutant allele results in decreased activity of the mutantcyclin SDS like protein compared to the wild type cyclin SDS likeprotein. The decreased activity is due to a knock-out of expression ofthe cyclin SDS like gene, a knock-down of expression of the gene, a lossof function of the encoded mutant cyclin SDS like protein or a decreaseof function of the mutant cyclin SDS like protein.

In one aspect, wherein the plant cell or plant is watermelon, the mutantallele of the cyclin SDS like protein is a mutant allele of the alleleencoding the protein of SEQ ID NO: 2 or a protein comprising substantialsequence identity to SEQ ID NO: 2, preferably at least 60%, 70%, 80%,90% sequence identity when the two full length sequences of thefunctional SDS like protein are pairwise aligned.

In another aspect, wherein the plant cell or plant is melon, the mutantallele of the cyclin SDS like protein is a mutant allele of the alleleencoding the protein of SEQ ID NO: 6 or a protein comprising substantialsequence identity to SEQ ID NO: 6, preferably at least 60%, 70%, 80%,90% sequence identity when the two full length sequences of thefunctional SDS like protein are pairwise aligned.

In another aspect, wherein the plant cell or plant is cucumber, themutant allele of the cyclin SDS like protein is a mutant allele of theallele encoding the protein of SEQ ID NO: 12 or a protein comprisingsubstantial sequence identity to SEQ ID NO: 12, preferably at least 60%,70%, 80%, 90% sequence identity when the two full length sequences ofthe functional SDS like protein are pairwise aligned.

In another aspect, wherein the plant cell or plant is tomato, the mutantallele of the cyclin SDS like protein is a mutant allele of the alleleencoding the protein of SEQ ID NO: 19 or a protein comprisingsubstantial sequence identity to SEQ ID NO: 19, preferably at least 60%,70%, 80%, 90% sequence identity when the two full length sequences ofthe functional SDS like protein are pairwise aligned.

In another aspect, wherein the plant cell or plant is pepper, the mutantallele of the cyclin SDS like protein is a mutant allele of the alleleencoding the protein of SEQ ID NO: 20 or a protein comprisingsubstantial sequence identity to SEQ ID NO: 20, preferably at least 60%,70%, 80%, 90% sequence identity when the two full length sequences ofthe functional SDS like protein are pairwise aligned.

In one aspect the watermelon, melon, cucumber, tomato or pepper plantcomprises the mutant cyclin SDS like allele in heterozygous form. Inanother aspect the watermelon, melon, cucumber, tomato or pepper plantcomprises the mutant cyclin SDS like allele in homozygous form, wherebythe plant produces seedless fruits upon pollination with own or otherpollen. In a preferred aspect the mutant cyclin SDS like allele is aknock out (i.e. the gene is not expressed) or the allele encodes anon-function cyclin SDS like protein.

Seeds from which such plants can be grown are encompassed herein as wellas the seedless fruits produced from said plants when the allele is inhomozygous form or the seeded fruits produced from said plants when theallele is in heterozygous form. Also any plant parts, such as cuttings,vegetative propagations, cells, etc. comprising at least one mutantcyclin SDS like allele in their genome are provided.

Also propagating and non-propagating cells comprising at least one copyof a mutant cyclin SDS like allele are provided herein. It is understoodthat such propagating or non-propagating cells can be part of a plantorgan or of an entire plant, or they can be isolated, e.g. in a cell ortissue culture.

The seed, plants and plant parts provided herein, comprising at leastone mutant cyclin SDS like allele in their genome, are preferablyagronomically useful plants, e.g. inbred lines, breeding lines,varieties or cultivars or F1 hybrids. Preferably they having goodagronomic characteristics, especially producing marketable fruits ofgood fruit quality and fruit uniformity.

As used herein, the term “variety” or “cultivar” means a plant groupingwithin a single botanical taxon of the lowest known rank, which can bedefined by the expression of the characteristics resulting from a givengenotype or combination of genotypes.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained fromcrossing two inbred parent lines. Thus, F1 hybrid seeds are seeds fromwhich F1 hybrid plants grow. F1 hybrids are more vigorous and higheryielding, due to heterosis. Inbred lines are essentially homozygous atmost loci in the genome.

A “plant line” or “breeding line” refers to a plant and its progeny. Asused herein, the term “inbred line” refers to a plant line which hasbeen repeatedly selfed and is nearly homozygous. Thus, an “inbred line”or “parent line” refers to a plant which has undergone severalgenerations (e.g. at least 5, 6, 7 or more) of inbreeding, resulting ina plant line with a high uniformity.

The watermelon cyclin SDS like allele is located on chromosome 7(between nucleotide 7450185 and 7445051) of the genome. The chromosomelocation can be determined by carrying out a BLAST against the wholegenome, e.g. on world wide web aticugi.org/pub/genome/watermelon/97103). The cucumber cyclin SDS likeallele appears also to be located on chromosome 5 (between nucleotides848447 and 852718) of the cucumber genome (world wide web aticugi.org/pub/genome/cucumber/Chinese_long/).

For watermelon, a mutant allele of a cyclin SDS like protein encodinggene can be obtained from the watermelon seeds being heterozygous orhomozygous for the mutant allele of the cyclin SDS like protein encodinggene, deposited under NCIMB 42532. A wild type allele of a cyclin SDSlike protein encoding gene can be obtained from the watermelon seedsbeing heterozygous or homozygous for the wild type cyclin SDS likeprotein encoding gene, deposited under NCIMB 42532. For the depositedseeds the respective allele of a cyclin SDS like protein encoding genewas designated emb1. Other mutant alleles of a cyclin SDS like proteinencoding gene can be generated de novo, e.g. by mutagenesis or by othermethods known to the skilled person. This applies for any plant species.

One example of generating another mutant SDS like allele de novo inwatermelon is provided in the Examples. Here, the inventors generated amutant population by mutagenizing watermelon seeds and then used TILLINGto identify a plant comprising a mutant SDS like allele. The identifiedallele comprises a single nucleotide replacement at nucleotide 1687 ofSEQ ID NO: 1, leading to a stop codon. The mutant allele thus encodes atruncated cyclin SDS like protein comprising only amino acids 1 to 223of the wild type protein (see SEQ ID NO: 18).

Plant cells, plant parts or plants or progeny thereofobtainable/obtained from seeds being heterozygous or homozygous for anallele of a SDS like protein encoding gene, deposited under NCIMB 42532are also an embodiment of the invention. In a preferred embodiment theplant cells, plant parts or plants or progeny thereof obtained fromseeds deposited under NCIMB 42532 are homozygous for a mutant allele ofa cyclin SDS like encoding gene. A further comprised embodiment of theinvention concerns plant cells, plant parts or plants, homozygous for amutant allele of a cyclin SDS like protein encoding geneobtained/obtainable after crossing a watermelon plant obtained fromseeds of deposit accession number NCIMB 42532 with another plant.Preferably the plant cells or plants obtained/obtainable after crossinga plant obtained from seeds of deposit accession number NCIMB 42532 withanother plant are subsequently self-pollinated and optionally in afurther step plants are selected which produce seedless fruits and/orplants are selected which are homozygous for a mutant allele of a cyclinSDS like encoding gene.

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus, all of which alleles relate to one trait orcharacteristic at a specific locus. In a diploid cell of an organism,alleles of a given gene are located at a specific location, or locus(loci plural) on a chromosome. One allele is present on each chromosomeof the pair of homologous chromosomes. A diploid plant species maycomprise a large number of different alleles at a particular locus.These may be identical alleles of the gene (homozygous) or two differentalleles (heterozygous).

“Wild type allele” refers herein to a version of a gene encoding a fullyfunctional protein (wild type protein). A sequence of a gene encoding afully functional cyclin SDS like protein is for example the codingsequence of the wild type cyclin SDS like protein sequences shown underSEQ ID NO 1 (from cultivated watermelon) and SEQ ID NO 5 (fromcultivated melon). The amino acid sequence encoded by this wild typecyclin SDS like protein encoding gene is depicted in SEQ ID NO 2 or SEQID NO 6, respectively. Other wild type cyclin SDS like proteins areshown under SEQ ID NO 12 (cucumber),

SEQ ID NO: 19 (tomato) and SEQ ID NO: 20 (pepper). Other cyclin SDS likeprotein encoding nucleic acid sequences encoding fully functional cyclinSDS like protein alleles (i.e. variant alleles, or allelic variants)exist in other plants and may comprise substantial sequence identitywith at least the coding sequence of the nucleic acid sequences shownunder SEQ ID NO 1 or SEQ ID NO 5 or with the amino acid sequences shownunder SEQ ID NO 2 or SEQ ID NO 6 or SEQ ID NO: 12, or SEQ ID NO: 19 orSEQ ID NO: 20. For example the cultivated cucumber cyclin SDS likeprotein of SEQ ID NO 12 has 86% amino acid sequence identity to the wildtype cyclin SDS like protein of melon (SEQ ID NO 6) and 70% amino acidsequence identity to the wild type SDS like protein of watermelon (SEQID NO 2).

A “mutant allele” is to be understood in connection with the presentinvention to mean an allele which has a mutation compared to thecorresponding wild type allele. An example of a mRNA transcribed from amutant allele of a cyclin SDS like protein encoding gene is shown underSEQ ID NO 3. The corresponding amino acid sequence encoded by the mRNAshown under SEQ ID NO 3 is shown under SEQ ID NO 4.

The term “locus” (loci plural) means a specific place or places or asite on a chromosome where for example a gene or genetic marker isfound.

A “mutation” in a nucleic acid molecule (DNA or RNA) is a change of oneor more nucleotides compared to the corresponding wild type sequence,e.g. by replacement, deletion or insertion of one or more nucleotides.Examples of such a mutation are point mutation, nonsense mutation,missense mutation, splice-site mutation, frame shift mutation or amutation in a regulatory sequence.

A “nucleic acid molecule” shall have the common understanding in theart. It is composed of nucleotides comprising either of the sugarsdeoxyribose (DNA) or ribose (RNA).

A “point mutation” is the replacement of a single nucleotide, or theinsertion or deletion of a single nucleotide.

A “nonsense mutation” is a (point) mutation in a nucleic acid sequenceencoding a protein, whereby a codon in a nucleic acid molecule ischanged into a stop codon. This results in a pre-mature stop codon beingpresent in the mRNA and results in translation of a truncated protein. Atruncated protein may have decreased function or loss of function.

A “missense or non-synonymous mutation” is a (point) mutation in anucleic acid sequence encoding a protein, whereby a codon is changed tocode for a different amino acid. The resulting protein may havedecreased function or loss of function.

A “splice-site mutation” is a mutation in a nucleic acid sequenceencoding a protein, whereby RNA splicing of the pre-mRNA is changed,resulting in an mRNA having a different nucleotide sequence and aprotein having a different amino acid sequence than the wild type. Theresulting protein may have decreased function or loss of function.

A “frame shift mutation” is a mutation in a nucleic acid sequenceencoding a protein by which the reading frame of the mRNA is changed,resulting in a different amino acid sequence. The resulting protein mayhave decreased function or loss of function.

A “deletion” in context of the invention shall mean that anywhere in agiven nucleic acid sequence at least one nucleotide is missing comparedto the nucleic sequence of the corresponding wild type sequence oranywhere in a given amino acid sequence at least one amino acid ismissing compared to the amino acid sequence of the corresponding (wildtype) sequence.

A “truncation” shall be understood to mean that at least one nucleotideat either the 3′-end or the 5′-end of the nucleotide sequence is missingcompared to the nucleic sequence of the corresponding wild type sequenceor that at least one amino acid at either the N-terminus or theC-terminus of the protein is missing compared to the amino acid sequenceof the corresponding wild type protein, whereby in a 3′-end orC-terminal truncation at least the first nucleotide at the 5′-end or thefirst amino acid at the N-terminus, respectively, is still present andin a 5′-end or N-terminal truncation at least the last nucleotide at the3′-end or the last amino acid at the C-terminus, respectively, is stillpresent. The 5′-end is determined by the ATG codon used as start codonin translation of a corresponding wild type nucleic acid sequence.

“Replacement” shall mean that at least one nucleotide in a nucleic acidsequence or one amino acid in a protein sequence is different comparedto the corresponding wild type nucleic acid sequence or thecorresponding wild type amino acid sequence, respectively, due to anexchange of a nucleotide in the coding sequence of the respectiveprotein.

“Insertion” shall mean that the nucleic acid sequence or the amino acidsequence of a protein comprises at least one additional nucleotide oramino acid compared to the corresponding wild type nucleic acid sequenceor the corresponding wild type amino acid sequence, respectively.

“Pre-mature stop codon” in context with the present invention means thata stop codon is present in a coding sequence (cds) which is closer tothe start codon at the 5′-end compared to the stop codon of acorresponding wild type coding sequence.

A “mutation in a regulatory sequence”, e.g. in a promoter or enhancer ofa gene, is a change of one or more nucleotides compared to the wild typesequence, e.g. by replacement, deletion or insertion of one or morenucleotides, leading for example to decreased or no mRNA transcript ofthe gene being made.

“Homozygous” is referred herein to mean that all copies of a given geneor allele at a corresponding chromosomal locus in a cell or an organismare identical. “Homozygous for a mutant allele” means that all copies ofthe respective mutant allele at the corresponding chromosomal locus in acell or an organism are identical.

“Heterozygous” is referred herein to mean that at least one copy of agiven gene or allele at a specific chromosomal locus in a cell or anorganism is different from the other copies of the gene(s) or allele(s)at the corresponding locus/loci in the other chromosome(s).“Heterozygous for a mutant allele” means that at least one allele at aspecific chromosomal locus in a cell or an organism has a differentsequence than the allele(s) at the corresponding locus/loci in the otherchromosome(s).

A “mutation in a protein” is a change of one or more amino acid residuescompared to the wild type sequence, e.g. by replacement, deletion,truncation or insertion of one or more amino acid residues.

Biotechnological methods for introducing mutations into a desiredgene/allele of a plant cell or plant are known in the art. Therefore,mutant alleles of a cyclin SDS like protein encoding gene can beproduced in plant cells or plants by using these methods. Examples forsuch technologies are in particular mutagenesis techniques or enzymeswhich induce double stranded DNA breaks (double stranded DNA breakinducing enzyme (DSBI)) in the genome of plants. Known and practisedtechnologies are rare-cleaving endonucleases and custom-tailoredrare-cleaving endonucleases including but not limited to homingendonucleases, also called meganucleases, transcription activator-likeeffectors fused to the catalytic domain of a nuclease (TALENs) andso-called CRISPR/Cas systems.

All these technologies are eligible for introducing a mutation intogenes in plant cells or plants. Therefore, plant cells and plantsaccording to the invention having a mutant allele of a cyclin SDS likeprotein encoding gene, wherein the mutation into the mutant allele wasintroduced by rare-cleaving endonucleases or custom-tailoredrare-cleaving endonucleases are also an embodiment of the invention.Concerning custom-tailored rare-cleaving endonucleases the mutation inthe mutant allele of a cyclin SDS like protein has preferably beenintroduced by a meganuclease, a TALENs or a CRISPR/Cas system.

As used herein, a “double stranded DNA break inducing enzyme (DSBI)” isan enzyme capable of inducing a double stranded DNA break at aparticular nucleotide sequence, called the “recognition site”.Rare-cleaving endonucleases are DSBI enzymes that have a recognitionsite of about 14 to 70 consecutive nucleotides, and therefore have avery low frequency of cleaving, even in larger genomes such as mostplant genomes.

“Homing endonucleases, also called meganucleases”, constitute a familyof such rare-cleaving endonucleases. They may be encoded by introns,independent genes or intervening sequences, and present strikingstructural and functional properties that distinguish them from the moreclassical restriction enzymes, usually from bacterialrestriction-modification Type II systems. Their recognition sites have ageneral asymmetry which contrasts the characteristic dyad symmetry ofmost restriction enzyme recognition sites. Several homing endonucleasesencoded by introns or inteins have been shown to promote the homing oftheir respective genetic elements into allelic intronless or inteinlesssites. By making a site-specific double strand break in the intronlessor inteinless alleles, these nucleases create recombinogenic ends, whichengage in a gene conversion process that duplicates the coding sequenceand leads to the insertion of an intron or an intervening sequence atthe DNA level.

A list of other rare cleaving meganucleases and their respectiverecognition sites is provided in Table I of WO 03/004659 (pages 17 to20) (incorporated herein by reference). These include I-Sce I, I-Chu I,I-Dmo I, I-Cre I, I-Csm I, PI-Fli I, Pt-Mtu I, I-Ceu I, I-Sce II, I-SceIII, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-Dhal, PI-Dra I,PI-May I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I,PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I,PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I,PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I or PI-Tsp I.

Furthermore, methods are available to design “custom-tailoredrare-cleaving endonucleases” that recognize basically any targetnucleotide sequence of choice. Briefly, chimeric restriction enzymes canbe prepared using hybrids between a zinc-finger domain designed torecognize a specific nucleotide sequence and the non-specificDNA-cleavage domain from a natural restriction enzyme, such as FokI.Such methods have been described e.g. in WO 03/080809, WO94/18313 orWO95/09233 and in Isalan et al., 2001, Nature Biotechnology 19, 656-660;Liu et al. 1997, Proc. Natl. Acad. Sci. USA 94, 5525-5530). Custom-mademeganucleases can be produced by selection from a library of variants,as described in WO2004/067736. Custom made meganucleases with alteredsequence specificity and DNA-binding affinity may also be obtainedthrough rational design as described in WO2007/047859.

Another example of custom-designed endonucleases include the so-called“TALE nucleases (TALENs)”, which are based on transcriptionactivator-like effectors (TALEs) from the bacterial genus Xanthomonasfused to the catalytic domain of a nuclease (e.g. FOKI). The DNA bindingspecificity of these TALEs is defined by repeat-variable diresidues(RVDs) of tandem-arranged 34/35-amino acid repeat units, such that oneRVD specifically recognizes one nucleotide in the target DNA. The repeatunits can be assembled to recognize basically any target sequences andfused to a catalytic domain of a nuclease create sequence specificendonucleases (see e.g. Boch et al., 2009, Science 326: p 1509-1512;Moscou and Bogdanove, 2009, Science 326: p 1501; Christian et al., 2010,Genetics 186: p 757-761; and WO10/079430, WO11/072246, WO2011/154393,WO11/146121, WO2012/001527, WO2012/093833, WO2012/104729, WO2012/138927,WO2012/138939). WO2012/138927 further describes monomeric (compact)TALENs and TALENs with various catalytic domains and combinationsthereof.

Recently, a new type of customizable endonuclease system has beendescribed; the so-called “CRISPR/Cas system”, which employs a specialRNA molecule (crRNA) conferring sequence specificity to guide thecleavage of an associated nuclease Cas9 (Jinek et al, 2012, Science 337:p 816-821). Such custom designed rare-cleaving endonucleases are alsoreferred to as non-naturally occurring rare-cleaving endonucleases.

A further method known in the art for introducing mutations into agene/allele of a plant cell or plant is the so-called “in vivomutagenesis”. Further discussion of the respective technology is givenherein below.

Plant cells or plants according to the invention having a mutant alleleof a cyclin SDS like protein encoding gene, wherein the mutation intothe mutant allele was introduced by in vivo mutagenesis are also anembodiment of the invention.

Various technologies commonly known in the art are suited to createinsertion mutations in plant cells or plants.

Further embodiments of the invention are plant cells and plantsaccording to the invention having a mutant allele of a cyclin SDS likeprotein encoding gene, wherein the mutation into the mutant allele wasintroduced by insertion mutagenesis.

The plant cells according to the invention and plants according to theinvention having a mutant allele of a cyclin SDS like protein encodinggene can be produced by so-called insertion mutagenesis. In particular,insertion of transposons and transfer DNA (T-DNA) sequences intogenes/alleles encoding cyclin SDS like proteins are suitable fordecreasing the expression and/or activity of the respectivegenes/alleles in which they are integrated (Thorneycroft et al., 2001,Journal of experimental Botany 52 (361), 1593-1601).

Additional discussion of the respective technologies known to a personskilled in the art will be provided herein further below.

“Insertion mutagenesis” is to be understood to mean particularly theinsertion of transposons or so-called transfer DNA (T-DNA) into a genecoding for a cyclin SDS like protein, whereby, as a result of which, theactivity of a cyclin SDS like protein in the cell concerned is decreasedor a non-functional cyclin SDS like protein is produced.

In a further preferred embodiment, the plants according to the inventionare male fertile plants.

The plants according to the invention are male fertile and produceseedless fruits when the mutant cyclin SDS like allele is present inhomozygous form. The advantage of male fertile plants over male sterileplants is that they will produce viable pollen and thus there is no needto plant a second, so called pollinator plant in the same field forinducing fruit set and development on the seedless fruit producingfemale plant. The whole area under cultivation thus can be planted withplants producing seedless fruits, leading to an increase of yield ofseedless fruits per area cultivated. Also, synchrony of flowering andfertilisation time is given for the male and female plant parts, becauseovules and pollen are produced by the same plant. This ensuressufficient pollination to take place for producing the most possibleamounts of fruits.

In context with the present invention, “male fertile plant” is to beunderstood to be a plant producing viable pollen. That viable or fertilepollen is produced can e.g. be shown by using the pollen from therespective plant for cross-pollinating another, different plant andobtaining viable seeds from this cross.

The seedless fruit producing phenotype of plants according to theinvention in one aspect is not only be generated in diploid plants butalso in polyploidy plants. In one aspect plants according to theinvention produce seedless fruits also when they have different degreeof ploidy. It is therefore well understood that the plant cells orplants according to the invention comprise plants having any degree ofploidy comprising plants with even numbered degree of ploidy (2n, 4n,6n, 8n etc.) and plants with uneven numbered degree of ploidy (3n, 5netc.). In one aspect, the mutant cyclin SDS like protein encoding geneis homozygous in diploid plants, but in another aspect the mutant SDSlike protein is homozygous in polyploidy plants, such as tetraploidwatermelons. “Homozygous in polyploidy plants” means that the locus oneach chromosome comprises the mutant allele and not the wild type alleleof the gene.

Polyploidisation is widespread in plants. It is responsible forincreasing genetic diversity and producing species showing increase inrobustness, size, vigour and disease resistance. Obvious advantages forpolyploid plants are heterosis and gene redundancy.

A number of today cultivated broad acre and plantation crops haveundergone one or more genome duplications. Examples are cotton(multiplication factor ×6), potato (×2, ×3), bread wheat (×3), oil seeds(×3), corn (×2), soybean (×2), sunflower (×2), banana (×2), apple (×2)and coffee (×2) (Renny-Byfield 7 Wendel, 2014, American J. Botany,101(10), 1711-1725).

In particular in vegetable breeding, polyploidy in various plants wasinduced by the use of chemicals including colchicine, colchamine,oryzalin, colcemid, trifluralin or amiprophosmethyl. Examples for genomeduplications in vegetables produced by the use of chemicals are diploidbrussels sprouts from haploid plants (2×), tetraploid peas (2×),tetraploid watermelons (2×), tetraploid muskmelons (2×), tetraploidonions (2×), octaploid cocoyams (4×), tetraploid snake gourds (2×),triploid and tetraploid fluted pumpkins (1.5×, 2×), tetraploid cucumbers(2×) and tetraploid french beans (2×) (Kazi, 2015, J. Global Biosciences4(3), 1774-1779).

Plants comprising a mutant allele of a cyclin SDS like protein encodinggene can be produced by various methods commonly known to a personskilled in the art. These methods comprise the use of seeds depositedunder accession number NCIMB 42532. A particular advantage of thesedeposited seeds is that the plants grown from the seeds which comprisethe mutant allele will be male fertile. Thus, the mutant allele of thecyclin SDS like protein encoding gene present in those plants can beintroduced into other plants by using the pollen of plants comprising amutant allele of the cyclin SDS like protein encoding gene forfertilising other plants, in particular other watermelon plants,especially cultivated watermelon. As the mutant allele of a cyclin SDSlike protein encoding gene is recessive, seedless fruit production isonly seen if no dominant wild type allele of a cyclin SDS like proteinencoding gene is present in the respective plant. Thus, in case a wildtype allele of a cyclin SDS like protein encoding gene is present inplants, these plants produce seeded fruits. When transferring a mutantallele of a cyclin SDS like protein encoding gene from a diploid seed,homozygous for a mutant allele of a cyclin SDS like protein encodinggene (e.g. from deposit accession number NCIMB 42532 or progeny thereof)to another plant which does not contain a recessive mutant allele of acyclin SDS like protein encoding gene, the F1 generation will beheterozygous and will not display the seedless fruit phenotype. The F1needs first to be self-pollinated for obtaining plants comprising theseedless fruit phenotype due to two copies (homozygous) of the recessivemutant allele being present.

A diploid plant comprising two copies of a mutant allele of a cyclin SDSlike protein encoding gene can be used to make a tetraploid plantcomprising four copies of a mutant allele of a cyclin SDS like proteinencoding gene. Such a tetraploid will have the same phenotype as thediploid, i.e. produce seedless fruits (which are tetraploid) and viablepollen.

When transferring a mutant allele of a cyclin SDS like protein encodinggene from a tetraploid plant to another tetraploid plant not comprisingthe recessive mutant allele of a cyclin SDS like protein encoding gene,the F1 generation will be heterozygous and does not display the seedlessfruit phenotype. Again the seedless fruit phenotype will only be seen inthe generation obtained from the self-pollinated F1 generation.

As mentioned, tetraploid plants comprising a seedless fruit phenotypecan be generated by duplicating the chromosomes of a diploid planthomozygous for mutant allele of a cyclin SDS like protein encoding gene(e.g. from seed, homozygous for a mutant allele of a cyclin SDS likeprotein encoding gene from deposit accession number NCIMB 42532 or fromplants, homozygous for a mutant allele of a cyclin SDS like proteinencoding gene obtained after crossing a plant obtained from seeds ofdeposit accession number NCIMB 42532 with another plant and optionallysubsequently self-pollinating the plants obtained from said cross). Thetetraploid plants so obtained comprise four copies of a mutant allele ofa cyclin SDS like protein encoding gene mutant allele.

It is commonly understood in the art that sexually reproducing cells ofplants (pollen and ovule) comprise a set of chromosomes which is half ofthe set of the remaining cells of said plant. Plant pollen and ovulescan be regenerated into whole plants. In case of plants having an evennumbered degree of ploidy it is therefore generally possible to reducethe degree of ploidy by half upon regeneration of pollen or ovules. Fromplants according to the invention having an even numbered degree ofploidy (e.g. 2n, 4n, 6n, 8n etc.) plants having a bisected set ofchromosomes (e.g. 1n, 2n, 3n, 4n, etc., respectively) can be produced bymeans of pollen or ovule regeneration.

Diploid plants according to the invention can e.g. be regenerated frompollen or ovule cells comprising a mutant allele of a cyclin SDS likeprotein encoding gene, the pollen or ovule cells being obtained from atetraploid plant comprising a mutant allele of a cyclin SDS like proteinencoding gene. Preferably, the pollen or ovule cells being obtained froma tetraploid plant comprise the mutant allele of a cyclin SDS likeprotein encoding gene in homozygous state. The derived diploid plantsmay then be used in further breeding and in generating plants having aseedless fruit phenotype.

Triploid plants can be produced by crossing a diploid (2n) plantaccording to the invention with a tetraploid (4n) plant according to theinvention. The hybrid plant seeds originating from said cross will betriploid (3n). Preferably the diploid (2n) and tetraploid (4n) plantsaccording to the invention crossed with each other both are homozygousfor a mutant allele of a cyclin SDS like protein encoding gene.

The resulting triploid seeds (and triploid plants grown from the seeds)will have three copies of the mutant allele. The diploid plant used forproducing a triploid hybrid can e.g. be plants obtained/obtainable fromseeds being homozygous for a mutant allele of a cyclin SDS like proteinencoding gene obtained from seeds of deposit accession number NCIMB42532.

Plants (e.g. diploid, triploid or tetraploid, or another ploidy) andplant parts (such as fruits) comprising a mutant allele of a cyclin SDSlike protein encoding gene obtainable/obtained by one of the methodsjust described are also an embodiment of the invention. Also seeds fromwhich such plants can be grown are an embodiment of the invention.

In a preferred embodiment of the invention the plant cells or plantsaccording to the invention have an even numbered degree of ploidy,preferably, they are diploid (2n) or tetraploid (4n).

Plants with an uneven numbered degree of ploidy, e.g. triploid (3n)plants are commonly male and female sterile, because during meiosis thechromosomes cannot be equally divided to the daughter cells. Theadvantage of plants with an even numbered degree of ploidy, e.g. diploid(2n) or tetraploid (4n) plants over plants with an uneven numbereddegree of ploidy, e.g. triploid (3n) plants is that plants with evennumbered degree of ploidy can produce viable pollen and/or viableovules. As a consequence plants with an even numbered degree of ploidycan be grown without the need of a second, different, so calledpollinator plant needed for inducing fruit set and development in theplant with uneven numbered degree of ploidy. Pollinator plants will alsoproduce fruits which commonly will be seed bearing (or seeded). Theseseed bearing fruits have to be separated from the seedless fruits uponor after harvesting. Thus, plants having an even numbered degree ofploidy have the advantage over plants with uneven numbered degree ofploidy that there is no need to separate undesired seed bearing fruitsproduced by pollinator plants from the desired seedless fruits.

“Even numbered degree of ploidy” in context of the present inventionmeans that the number of homologous chromosome sets present in a cell ororganism when divided by two results in an integer. The cells ororganisms thus are diploid (2n), tetraploid (4n), hexaploid (6n),octaploid (8n) etc.

“Uneven numbered degree of ploidy” in context of the present inventionmeans that the number of homologous chromosome sets present in a cell ororganism when divided by two does not result an integer. The cells ororganisms thus are haploid (1n), triploid (3n) etc.

“Diploid plant cell or plant” in context of the present invention meansa plant, vegetative plant part(s), fruit or seed or plant cell, havingtwo sets of corresponding chromosomes, designated herein as 2n.

“Tetraploid cell or plant” in context of the present invention means aplant, vegetative plant part(s), fruit or seed or plant cell, havingfour sets of corresponding chromosomes, designated herein as 4n.

The plant cells according to the invention can be those plant cellswhich can be regenerated into a whole plant or those which cannot beregenerated into whole plants. Thus, the plant cells according to theinvention may be those plant cells which are not eligible to regeneratea whole plant.

In a preferred embodiment the plants according to the invention are malefertile and have an even numbered degree of ploidy. Preferably, plantsaccording to the invention are male fertile and are diploid (2n) ortetraploid (4n).

In another preferred embodiment, the plants according to the inventionare stenospermocarpic plants. More preferably the plants according tothe invention are male fertile stenospermocarpic plants. Even furtherpreferred the plants according to the invention are male fertile,stenospermocarpic and have an even numbered degree of ploidy. Inparticular preferred are plants according to the invention which aremale fertile, stenospermocarpic, diploid (2n) or tetraploid (4).

Stenospermocarpic plants produce seedless fruits. Male fertilestenospermocarpic plants have the advantage over known stenospermocarpicplants that they do not need a different pollinator plant grown in thesame area but that they nevertheless produce seedless fruits. Pollinatorplants will produce undesired seed bearing fruits, which will have to beseparated from the seedless fruits. Thus, stenospermocarpic male fertileplants have the advantage that there is no competition for growing spaceand nutrients between a plant producing the desired seedless fruits andthe polliniser plants, which increases yield of the desired seedlessfruits per planting area available.

“Stenospermocarpy” is generally understood in the art and also to beunderstood in connection with the present invention to mean thatinduction of fruit set and development requires pollination but withoutthe fruits producing mature or viable seeds. Mature or viable seeds arenot developed in stenospermocarpic plants due to arrested seeddevelopment or degradation of ovules and/or embryos and/or endosperm orabortion of the ovules and/or embryos and/or endosperm before maturityis reached.

To be differentiated from stenospermocarpy is parthenocarpy.“Parthenocarpy” is generally understood in the art and also to beunderstood in connection with the present invention to describe thedevelopment of fruits without fertilization of the female ovule. Apollination process is not needed for producing fruits which fruitshowever as a consequence of the lack of pollination are seedless.

In a further preferred embodiment of the invention, the plants accordingto the invention produce seedless fruits.

The fruits of plants according to the invention may contain structureswhich have a seed like appearance. These structures having a seed likeappearance are normally white and soft compared to the seeds of wildtype plants which are dark brown or black and hard. The structureshaving a seed like appearance in fruits of plant according to theinvention are denoted sometimes as empty seeds. However, they are notrue seeds, because they do not comprise a viable embryo but arestructures originating from the ovule integument.

In a more preferred embodiment of the invention the plants according tothe invention produce seedless fruits and/or are male fertile and/orhave an even degree of ploidy and/or are stenospermocarpic. Even morepreferred the plants according to the invention produce seedless fruits,are male fertile, are diploid (2n) or tetraploid (4n) and arestenospermocarpic.

The term “fruit” in its botanical meaning is commonly understood to be aseed bearing structure developed from the ovary of angiosperm flowers.

A “seedless fruit” as commonly used in the art and in particular inbreeding, although being somehow contradicting the botanical meaning of“fruit”, is to be understood in context with the present invention to bea fruit without mature or viable seeds. Mature or viable seeds can begerminated in soil under conditions appropriate for the respective plantand grown into plants. This test can be used to determine if a plantproduces seedless fruits. Seedless fruits will not produce seed whichwill germinate and grow into a plant under conditions appropriate forthe respective plant.

By knowing the causative gene for the production of seedless fruitsdisclosed herein, it is now possible to produce seedless fruit producingplants by various known methods. These methods can rely on producing andselecting plants having mutant alleles encoding non-functional cyclinSDS like proteins or mutant alleles encoding cyclin SDS like proteinswith decreased or loss of function or by using conventional mutationagents like chemicals, high energy radiation (e.g. x-rays, neutronradiation, gamma radiation or UV radiation). It is also possible bymeans of gene technology to produce plants having non-functional cyclinSDS like proteins or having decreased activity of cyclin SDS likeproteins.

Plants according to the invention can be produced by introducing one ormore mutations into an allele of a cyclin SDS like protein encodinggene.

A further embodiment of the present invention therefore concerns amethod for production of a plant comprising the steps of

-   a) introducing mutations in a population of plants-   b) selecting a male fertile plant producing seedless fruits-   c) verifying if the plant selected under b) has a mutation in an    allele of a cyclin SDS like protein encoding gene, optionally-   d) growing/cultivating the plants obtained under c).

Thus one aspect is a method for production of a plant comprising thesteps of

-   -   a) introducing mutations in a population of plants    -   b) selecting a male fertile plant producing seedless fruit    -   c) verifying if the plant selected under b) has a mutation in an        allele encoding a cyclin SDS like protein encoding gene and        selecting a plant comprising such a mutation, and optionally    -   d) growing/cultivating the plants obtained under c), wherein the        wild type allele of the gene encodes a cyclin SDS like protein        comprising at least 60% sequence identity to any one of the        proteins selected from the group of: SEQ ID NO 2 or SEQ ID NO 6        or SEQ ID NO 12 or SEQ ID NO 19 or SEQ ID NO 20.

However, in one aspect the order of the steps can also be different.

So in one aspect a method for production of a plant comprising the stepsof

-   -   a) introducing mutations in a population of plants    -   b) identifying a plant which has a mutation in an allele        encoding a cyclin SDS like protein encoding gene and optionally    -   c) determining whether the plant is male fertile and whether the        plant, or a progeny plant produced by self-fertilization,        produces seedless fruits.

Optionally, the method comprises selecting a plant comprising at leastone copy of a mutant allele of a gene encoding a cyclin SDS likeprotein. The mutant allele, when in homozygous form, results in theproduction of seedless fruits. The plant comprising the allele is malefertile.

In one aspect the wild type allele of the gene encodes a cyclin SDS likeprotein comprising at least 60% sequence identity to any one of theproteins selected from the group of: SEQ ID NO 2 or SEQ ID NO 6 or SEQID NO 12 or SEQ ID NO 19 or SEQ ID NO 20.

Also, step a) of these methods (i.e. introducing mutations in apopulation of plants) can also be omitted in the above methods.

“Population of plants” shall mean in context with the present inventionmore than one whole plant and shall comprise also plant parts, fruits,seeds or plant cells. The plant parts, fruits, seeds or plant cells ineach case originate from more than one plant meaning that concerning a“population of plant parts, fruits, seeds or plant cells”, the plantparts, fruits, seeds or plant cells, respectively, are not obtained froma single plant but from a plurality of plants.

Chemical substances, which can be used to produce chemically inducedmutations, and the mutations resulting from the effect of thecorresponding mutagens are, for example described in Ehrenberg andHusain, 1981, (Mutation Research 86, 1-113), Müller, 1972 (BiologischesZentralblatt 91 (1), 31-48). The production of rice mutants using gammaradiation, ethyl methane sulphonate (EMS), N-methyl-N-nitrosurea orsodium azide (NaN₃) is described, for example, in Jauhar and Siddiq(1999, Indian Journal of Genetics, 59 (1), 23-28), in Rao (1977,Cytologica 42, 443-450), Gupta and Sharma (1990, Oryza 27, 217-219) andSatoh and Omura (1981, Japanese Journal of Breeding 31 (3), 316-326).The production of wheat mutants using NaN₃ or maleic hydrazide isdescribed in Arora et al. (1992, Annals of Biology 8 (1), 65-69). Anoverview of the production of wheat mutants using different types ofenergy-rich radiation and chemical substances is presented inScarascia-Mugnozza et al. (1993, Mutation Breeding Review 10, 1-28).Svec et al. (1998, Cereal Research Communications 26 (4), 391-396)describes the use of N-ethyl-N-nitrosurea for producing mutations intriticale. The use of MMS (methyl methane sulphonic acid) and gammaradiation for the production of millet mutants is described inShashidhara et al. (1990, Journal of Maharashtra AgriculturalUniversities 15 (1), 20-23).

The manufacture of mutants in plant species, which mainly propagatevegetatively, has been described, for example, for potatoes, whichproduce a modified starch (Hovenkamp-Hermelink et al. (1987, Theoreticaland Applied Genetics 75, 217-221) and for mint with increased oil yieldor modified oil quality (Dwivedi et al., 2000, Journal of Medicinal andAromatic Plant Sciences 22, 460-463).

All these methods are basically suitable in the method for production ofa plant according to the invention for producing mutant alleles in genesencoding a cyclin SDS like protein. In the method for production of aplant according to the invention preferably the mutant population isproduced by applying ethyl methane sulphonate (EMS) to plants or seedsof plants for introducing mutations.

Selecting plants producing seedless fruits can be done by simply visiblescreening/phenotyping the fruits. As the phenotype of seedlessness isonly seen in homozygous condition, selfing of the population ofmutagenized plants is preferred before phenotyping. That fertile pollenis produced by a plant can e.g. be shown by using the pollen from therespective plant for cross-pollinating another, different, femalefertile plant. In case the seeds from this cross are viable the pollenused in the cross-pollinations was fertile. Mutations in the appropriatealleles, in particular in alleles of cyclin SDS like protein encodinggenes, can be found with the help of methods known to the person skilledin the art. In particular, analyses based on hybridisations with probes(Southern Blot), amplification by means of polymerase chain reaction(PCR), sequencing of related genomic sequences and the search forindividual nucleotide exchanges can be used for this purpose. A methodof identifying mutations based on hybridisation patterns is, forexample, the search for restriction fragment length differences(Restriction Fragment Length Polymorphism, RFLP) (Nam et al., 1989, ThePlant Cell 1, 699-705; Leister and Dean, 1993, The Plant Journal 4 (4),745-750). A method based on PCR is, for example, the analysis ofamplified fragment length differences (Amplified Fragment LengthPolymorphism, AFLP) (Castiglioni et al., 1998, Genetics 149, 2039-2056;Meksem et al., 2001, Molecular Genetics and Genomics 265, 207-214; Meyeret al., 1998, Molecular and General Genetics 259, 150-160). The use ofamplified fragments excised with restriction endonucleases (CleavedAmplified Polymorphic Sequences, CAPS) can also be used upon for theidentification of mutations (Konieczny and Ausubel, 1993, The PlantJournal 4, 403-410; Jarvis et al., 1994, Plant Molecular Biology 24,685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753). Methodsfor the determination of SNPs have been described by Qi et al. (2001,Nucleic Acids Research 29 (22), e116) Drenkard et al. (2000, PlantPhysiology 124, 1483-1492) and Cho et al. (1999, Nature Genetics 23,203-207) amongst others. Methods, which allow several plants to beinvestigated for mutations in certain genes in a short time, areparticularly suitable. Such a method, so-called TILLING (TargetingInduced Local Lesions IN Genomes), has been described by McCallum et al.(2000, Plant Physiology 123, 439-442).

It is well known in the art, that today also other methods are usefulfor identifying plant cells according to the invention and plantsaccording to the invention having a mutant allele of a cyclin SDS likeprotein encoding gene. These methods comprise e.g. so-called forwardscreening approaches. In the forward screening approaches a mutantpopulation is produced. Plants of the mutant population, e.g. M2 plantsare screened for seedless fruit producing plants, which are then crossedto various different inbred lines for producing a mapping population.The mapping population is then analysed by methods well known in the artto identify the allele causing the seedless fruit phenotype. Othermethods for identifying if a plant cell or plant comprises a mutantallele of a cyclin SDS like protein encoding gene comprise sequencing ofthe respective alleles and SNP marker analyses with methods common inthe art and e.g. discussed in Thomson (2014, Plant Breeding andBiotechnology 2, 195-212).

These methods are basically suitable for identifying plant cellsaccording to the invention and plants according to the invention havinga mutant allele of a cyclin SDS like protein encoding gene.

Growing the male fertile, seedless fruit producing plants having amutant allele of a cyclin SDS like protein encoding gene identified inthe method for production of a plant according to the invention can bedone by conventional methods in a greenhouse or in the field.Cultivation and/or propagation of these plants can be done by methodscommon in the art like e.g. by cuttings, in vitro tissue, cell,protoplast, embryo or callus cultures or micropropagation or by graftingcuttings to a different rootstock.

In a preferred embodiment of the invention, the methods for productionof a plant according to the invention are used for producing plantsaccording to the invention. The preferred embodiments described abovefor plants according to the invention are accordingly applicable to themethods for production of a plant according to the invention.

Plants obtainable/obtained by the method for production of a plantaccording to the invention are also an embodiment of the invention.

Various methods available in gene technology offer further possibilitiesto produce plants having mutant alleles of a cyclin SDS like proteinencoding gene or having non-functional cyclin SDS like proteins orhaving decreased activity of cyclin SDS like proteins or showing adecreased expression of cyclin SDS like proteins.

All these methods are based on the introduction of a foreign or ofseveral foreign nucleic acid molecules into the genome of plant cells orplants and therefore are basically suitable for producing plant cellsaccording to the invention and plants according to the invention.

A further embodiment of the invention therefore concerns a method forproduction of a plant comprising the steps of

-   a) introduction of a foreign nucleic acid molecule into a plant,    wherein the foreign nucleic acid molecule is chosen from the group    consisting of    -   i) DNA molecules, which code at least one antisense RNA, which        effects a reduction in the expression of an endogenous gene        encoding a cyclin SDS like protein;    -   ii) DNA molecules, which by means of a co-suppression effect        lead to the reduction in the expression of an endogenous gene        encoding a cyclin SDS like protein;    -   iii) DNA molecules, which code at least one ribozyme, which        splits specific transcripts of an endogenous gene encoding a        cyclin SDS like protein;    -   iv) DNA molecules, which simultaneously code at least one        antisense RNA and at least one sense RNA, wherein the said        antisense RNA and the said sense RNA form a double-stranded RNA        molecule, which effects a reduction in the expression of an        endogenous gene encoding a cyclin SDS like protein having (RNAi        technology);    -   v) nucleic acid molecules introduced by means of in vivo        mutagenesis, which lead to a mutation or an insertion of a        heterologous sequence in an endogenous gene encoding a cyclin        SDS like protein, wherein the mutation or insertion effects a        reduction in the expression of a gene encoding a cyclin SDS like        protein or results in the synthesis of an inactive cyclin SDS        like protein;    -   vi) nucleic acid molecules, which code an antibody, wherein the        antibody results in a decrease in the activity of an endogenous        gene encoding a cyclin SDS like protein due to the bonding of        the antibody to an endogenous cyclin SDS like protein,    -   vii) DNA molecules, which contain transposons, wherein the        integration of these transposons leads to a mutation or an        insertion in an endogenous gene encoding a cyclin SDS like        protein, which effects a reduction in the expression of an        endogenous gene encoding a cyclin SDS like protein, or results        in the synthesis of an inactive cyclin SDS like protein;    -   viii) T-DNA molecules, which, due to insertion in an endogenous        gene encoding a cyclin SDS like protein, effect a reduction in        the expression of an endogenous gene encoding a cyclin SDS like        protein, or result in the synthesis of an inactive cyclin SDS        like protein, and/or    -   ix) nucleic acid molecules encoding rare-cleaving endonucleases        or custom-tailored rare-cleaving endonucleases preferably a        meganuclease, a TALENs or a CRISPR/Cas system-   b) selecting a plant producing seedless fruits, optionally-   c) verifying if the plant selected under b) has a decreased activity    of a cyclin SDS like protein compared to wild type plants into whose    genome no foreign nucleic acid molecules had been integrated, and    optionally-   d) growing/cultivating the plants obtained under c).

In a preferred embodiment of the invention, the method according to theinvention comprising introducing a foreign nucleic acid molecule into aplant cell concerns a method for the production of a male fertile plant,meaning that selection of a plant being male fertile and producingseedless fruit takes place (in step b and/or c).

The decrease of the activity of cyclin SDS like proteins in plant cellsor plants according to the invention or in the method according to theinvention comprising introducing a foreign nucleic acid molecule into aplant cell can be brought about by expression of antisense orco-suppression constructs.

For inhibiting the expression of genes by means of antisense orco-suppression technology, a DNA molecule can be used, for example,which includes the whole coding sequence for a cyclin SDS like protein,including any existing flanking sequences, as well as DNA molecules,which include only parts of the coding sequence, whereby these partsmust be long enough to produce an antisense effect or a co-suppressioneffect respectively in the cells. In general, sequences up to a minimumlength of 20 bp or 21 bp (or nucleotides), preferably a minimum lengthof at least 100 bp (or nucleotides), particularly preferably of at least500 bp (or nucleotides) are suitable. For example, the DNA moleculeshave a length of 21-100 bp (or nucleotides), preferably of 100-500 bp(or nucleotides), particularly preferably over 500 bp (or nucleotides).

The use of DNA sequences, which have a high degree of identity with theendogenous sequences occurring in the plant cell and which encode cyclinSDS like proteins, is also suitable for antisense or co-suppressionpreparations. The minimum identity should be greater than ca. 65%,preferably greater than 80%. The use of sequences with identities of atleast 90%, in particular between 95% and 100%, is to be preferred. Themeaning of the term “sequence identity” is defined elsewhere herein.

Furthermore, the use of introns, i.e. of non-coding areas of genes,which code for cyclin SDS like proteins, is also conceivable forachieving an antisense or a co-suppression effect. The use of intronsequences for inhibiting the gene expression of genes, which code forstarch biosynthesis proteins, has e.g. been described in theinternational patent applications WO97/04112, WO97/04113, WO98/37213,WO98/37214.

The person skilled in the art knows how to achieve an antisense and aco-suppression effect. For example, the method of co-suppressioninhibition has been described in Jorgensen (Trends Biotechnol. 8 (1990),340-344), Niebel et al., (Curr. Top. Microbiol. Immunol. 197 (1995),91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995),43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159),Vaucheret et al., (Mol. Gen. Genet. 248 (1995), 311-317), de Borne etal. (Mol. Gen. Genet. 243 (1994), 613-621).

The expression of ribozymes for reducing the activity of particularenzymes in cells is also known to the person skilled in the art, and isdescribed, for example, in EP-B1 0321201. The expression of ribozymes inplant cells has been described, for example, in Feyter et al. (Mol. Gen.Genet. 250, (1996), 329-338).

The decrease of the activity of cyclin SDS like proteins in plant cellsor plants according to the invention or in the method according to theinvention comprising introducing a foreign nucleic acid molecule into aplant cell can also be brought about by the simultaneous expression ofsense and antisense RNA molecules (RNAi technology) of the respectivetarget gene to be repressed, preferably of the cyclin SDS like proteinencoding gene.

This can be achieved, for example, by the use of chimeric constructs,which contain “inverted repeats” of the respective target gene or partsof the target gene. In this case, the generic constructs code for senseand antisense RNA molecules of the respective target gene. Sense andantisense RNA are synthesized simultaneously in planta as an RNAmolecule, wherein sense and antisense RNA are separated from one anotherby a spacer, and are able to form a double-stranded RNA molecule.

It has been shown that the introduction of inverted repeat DNAconstructs into the genome of plant cells or plants is a very effectivemethod of repressing the genes corresponding to the inverted repeat DNAconstructs (Waterhouse et al., Proc. Natl. Acad. Sci. USA 95, (1998),13959-13964; Wang and Waterhouse, Plant Mol. Biol. 43, (2000), 67-82;Singh et al., Biochemical Society Transactions Vol. 28 part 6 (2000),925-927; Liu et al., Biochemical Society Transactions Vol. 28 part 6(2000), 927-929); Smith et al., (Nature 407, (2000), 319-320;international patent application WO99/53050 A1). Sense and antisensesequences of the target gene or of the target genes can also beexpressed separately from one another by means of similar or differentpromoters (Nap, J-P et al, 6^(th) International Congress of PlantMolecular Biology, Quebec, 18th-24th June, 2000; Poster S7-27,Presentation Session S7). The decrease of the activity of cyclin SDSlike proteins in plant cells according to the invention or plantsaccording to the invention can therefore also be achieved by producingdouble-stranded RNA molecules. In this regard, “inverted repeats” of DNAmolecules of cyclin SDS like protein encoding genes or cDNAs arepreferably introduced into the genome of plants, wherein the DNAmolecules (cyclin SDS like protein encoding gene or cDNA or fragments ofthese genes or cDNAs) to be transcribed are under the control of apromoter, which controls the expression of said DNA molecules.

Fragments of any of the nucleic acid molecules encoding cyclin SDS likeproteins are therefore also an aspect of the invention. Such fragmentshave various uses, e.g. as primers or probes, or they can beincorporated into transformation vectors and used to generate plantsproducing seedless fruits.

Such fragments of nucleic acid molecules encoding cyclin SDS likeproteins may be of various sizes, e.g. at least 10 nucleotides, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 200, at least 300, atleast 400, at least 500 nucleotides or more.

In addition to this, it is known that the formation of double-strandedRNA molecules from promoter DNA molecules in plants in trans can lead tomethylation and transcriptional inactivation of homologous copies ofthese promoters, which are to be referred to in the following as targetpromoters (Mette et al., EMBO J. 19, (2000), 5194-5201). It is thereforepossible to reduce the gene expression of a particular target gene (e.g.cyclin SDS like protein encoding gene), which is naturally under thecontrol of this target promoter, by deactivating the target promoter.This means that, in this case, the DNA molecules, which include thetarget promoters of the genes to be repressed (target genes), incontrast to the original function of promoters in plants, are not usedas control elements for the expression of genes or cDNAs, but arethemselves used as transcribable DNA molecules.

For the production of double-stranded target promoter RNA molecules inplanta, which can occur there as RNA hairpin molecules, constructs arepreferably used, which contain the “inverted repeats” of the targetpromoter DNA molecules, wherein the target promoter DNA molecules areunder the control of a promoter, which controls the gene expression ofsaid target promoter DNA molecules. These constructs are subsequentlyintroduced into the genome of plants. The expression of the “invertedrepeats” of said target promoter DNA molecules in planta leads to theformation of double-stranded target promoter RNA molecules (Mette etal., EMBO J. 19, (2000), 5194-5201). The target promoter can beinactivated by this means. The decrease of the activity of cyclin SDSlike proteins in plant cells according to the invention and plantsaccording to the invention can therefore also be achieved by theintroduction of double-stranded RNA molecules of promoter sequences ofcyclin SDS like protein encoding genes into plant cells or plants. Inthis regard, “inverted repeats” of promoter DNA molecules of cyclin SDSlike protein encoding genes are preferably introduced into the genome ofplants, wherein the target promoter DNA molecules (promoter of a cyclinSDS like protein encoding gene) to be transcribed are under the controlof a promoter, which controls the expression of said target promoter DNAmolecules.

For inhibiting the expression of genes by means of the simultaneousexpression of sense and antisense RNA molecules (RNAi technology), a DNAmolecule can be used, for example, which includes the whole codingsequence for a cyclin SDS like protein, including any existing flankingsequences, as well as DNA molecules, which include only parts of thecoding sequence, whereby these parts must be long enough to produce aso-called RNAi effect in the cells. The parts of the cyclin SDS likeprotein encoding gene can be chosen from coding sequences,non-translated down- or up-stream sequences, introns, promoters and/orenhancers. In general, sequences with a minimum length of 20 bp (ornucleotides), preferably a minimum length of at least 25 bp (ornucleotides), particularly preferably of at least 50 bp (or nucleotides)are suitable. For example, the DNA molecules have a length of 20 to 25bp (or nucleotides), preferably of 26 to 50 bp (or nucleotides),particularly preferably greater than 50 bp (or nucleotides).

The use of DNA sequences, which have a high degree of identity with theendogenous sequences occurring in the plant cells and which code acyclin SDS like protein, is also suitable for the simultaneousexpression of sense and antisense RNA molecules (RNAi technology). Theminimum identity should be greater than ca. 65%, preferably greater than80%. The use of sequences with identities of at least 90%, in particularbetween 95% and 100%, is to be particularly preferred. Sequences, whichcontain successive stretches of nucleic acids sequences comprised by thenucleic acid sequence shown under SEQ ID NO 1, are particularly suitablefor inhibiting cyclin SDS like protein encoding genes by means of RNAitechnology.

The decrease of the activity of cyclin SDS like proteins in plant cellsaccording to the invention and plants according to the invention or inthe method according to the invention comprising introducing a foreignnucleic acid molecule into a plant cell can be achieved by so-called “invivo mutagenesis”, in which a hybrid RNA-DNA oligonucleotide(“Chimeroplast”) is introduced into plant cells (Kipp, P. B. et al.,Poster Session at the “5^(th) International Congress of Plant MolecularBiology, 21st-27th September 1997, Singapore; R. A. Dixon and C. J.Arntzen, meeting report on “Metabolic Engineering in Transgenic Plants”,Keystone Symposia, Copper Mountain, Colo., USA, TIBTECH 15, (1997),441-447; international patent application WO 9515972; Kren et al.,Hepatology 25, (1997), 1462-1468; Cole-Strauss et al., Science 273,(1996), 1386-1389; Beetham et al., 1999, PNAS 96, 8774-8778).

A part of the DNA components of the RNA-DNA oligonucleotide ishomologous to a nucleic acid sequence of an endogenous cyclin SDS likeprotein encoding gene, but, in comparison with the nucleic acid sequenceof an endogenous cyclin SDS like protein encoding gene, it has amutation or contains a heterologous region, which is surrounded by thehomologous regions. By base pairing of the homologous regions of theRNA-DNA oligonucleotide and the endogenous nucleic acid moleculefollowed by homologous recombination, the mutation or heterologousregion contained in the DNA components of the RNA-DNA oligonucleotidecan be transferred into the genome of a plant cell. This leads to adecrease of the activity of one or more cyclin SDS like proteins.

The decrease of the activity of cyclin SDS like proteins in plant cellsaccording to the invention and plants according to the invention or inthe method according to the invention comprising introducing a foreignnucleic acid molecule into a plant cell can be achieved by introducingnucleic acid molecules encoding antagonists/inhibitors of cyclin SDSlike proteins into a plant cell. The person skilled in the art knowsthat he can achieve a decrease of the activity of cyclin SDS likeproteins by the expression of non-functional derivatives, in particulartrans-dominant mutations, of such proteins, and/or by the expression ofantagonists/inhibitors of such proteins. Antagonist/inhibitors of suchproteins include, for example, antibodies, antibody fragments ormolecules with similar bonding characteristics. For example, acytoplasmatic scFv antibody has been used to modulate the activity ofthe phytochrome A protein in genetically modified tobacco plants (Owen,Bio/Technology 10 (1992), 790-4; Review: Franken, E, Teuschel, U. andHain, R., Current Opinion in Biotechnology 8, (1997), 411-416; Whitelam,Trends Plant Sci. 1 (1996), 268-272; Conrad and Manteufel, Trends inPlant Science 6, (2001), 399-402; De Jaeger et al., Plant MolecularBiology 43, (2000), 419-428). The decrease of the activity of abranching enzyme in potato plants by expressing a specific antibody hasbeen described by Jobling et al. (Nature Biotechnology 21, (2003),77-80). Here, the antibody was provided with a plastid target sequenceso that the inhibition of proteins localised in plastids was guaranteed.

The decrease of the activity of cyclin SDS like proteins in plant cellsaccording to the invention and plants according to the invention or inthe method according to the invention comprising introducing a foreignnucleic acid molecule into a plant cell can be achieved by introducingnucleic acid molecules comprising transposon sequences into the plantcell. Insertion of transposon sequences into the sequence of anendogenous cyclin SDS like protein encoding gene, will effect areduction in the expression of an endogenous cyclin SDS like protein.

The transposons can be endogenous transposons (homologous to the plant)and also those that do not occur naturally in said cell (heterologous tothe plant) but in each case have to be introduced into a plant cell orplant by means of genetic engineering methods, such as transformation ofa cell, for example

Changing the expression of genes by means of transposons is known to theperson skilled in the art. An overview of the use of endogenous andheterologous transposons as tools in plant biotechnology is presented inRamachandran and Sundaresan (2001, Plant Physiology and Biochemistry 39,234-252). The possibility of identifying mutations in which specificgenes have been inactivated by transposon insertion mutagenesis ispresented in an overview by Maes et al. (1999, Trends in Plant Science 4(3), 90-96). The production of rice mutants with the help of endogenoustransposons is described by Hirochika (2001, Current Opinion in PlantBiology 4, 118-122). The identification of maize genes with the help ofendogenous retrotransposons is presented, for example, by Hanley et al.(2000, The Plant Journal 22 (4), 557-566). The possibility ofmanufacturing mutants with the help of retrotransposons and methods ofidentifying mutants are described by Kumar and Hirochika (2001, Trendsin Plant Science 6 (3), 127-134). The activity of technological(artificial) transposons in different species has been described bothfor dicotyledonous and for monocotyledonous plants: e.g. for rice (Grecoet al., 2001, Plant Physiology 125, 1175-1177; Liu et al., 1999,Molecular and General Genetics 262, 413-420; Hiroyuki et al., 1999, ThePlant Journal 19 (5), 605-613; Jeon und Gynheung, 2001, Plant Science161, 211-219), barley (2000, Koprek et al., The Plant Journal 24 (2),253-263) Arabidopsis thaliana (Aarts et al., 1993, Nature 363, 715-717,Schmidt und Willmitzer, 1989, Molecular and General Genetics 220, 17-24;Altmann et al., 1992, Theoretical and Applied Genetics 84, 371-383;Tissier et al., 1999, The Plant Cell 11, 1841-1852), tomato (Belzile undYoder, 1992, The Plant Journal 2 (2), 173-179) and potato (Frey et al.,1989, Molecular and General Genetics 217, 172-177; Knapp et al., 1988,Molecular and General Genetics 213, 285-290).

Basically, the plant cells according to the invention and the plantsaccording to the invention can be produced both with the help ofhomologous and heterologous transposons.

In conjunction with the present invention, plant cells and plantsaccording to the invention can also be produced by the use of so-calledinsertion mutagenesis (overview article: Thorneycroft et al., 2001,Journal of experimental Botany 52 (361), 1593-1601). The decrease of theactivity of cyclin SDS like proteins in plant cells according to theinvention and plants according to the invention or in the methodaccording to the invention comprising introducing a foreign nucleic acidmolecule into a plant cell can be achieved by introducing nucleic acidmolecules comprising T-DNA sequences into the plant cell. T-DNAinsertion mutagenesis is based on the fact that certain sections (T-DNA)of Ti plasmids from Agrobacterium can integrate into the genome of plantcells. The place of integration in the plant chromosome is not defined,but can take place at any point. If the T-DNA integrates into a part ofthe chromosome, which constitutes a gene function, then this can lead toa change in the gene expression and thus also to a change in theactivity of a protein coded by the gene concerned. In particular, theintegration of a T-DNA into the coding area of a protein often leads tothe corresponding protein no longer being able to be synthesized at all,or no longer synthesized in active form, by the cell concerned. The useof T-DNA insertions for producing mutants is described, for example, forArabidopsis thaliana (Krysan et al., 1999, The Plant Cell 11, 2283-2290;Atipiroz-Leehan and Feldmann, 1997, Trends in genetics 13 (4), 152-156;Parinov and Sundaresan, 2000, Current Opinion in Biotechnology 11,157-161) and rice (Jeon and An, 2001, Plant Science 161, 211-219; Jeonet al., 2000, The Plant Journal 22 (6), 561-570). Methods foridentifying mutants, which have been produced with the help of T-DNAinsertion mutagenesis, are described, amongst others, by Young et al.,(2001, Plant Physiology 125, 513-518), Parinov et al. (1999, The Plantcell 11, 2263-2270), Thorneycroft et al. (2001, Journal of ExperimentalBotany 52, 1593-1601), and McKinney et al. (1995, The Plant Journal 8(4), 613-622).

T-DNA insertion mutants have been produced in great numbers forArabidopsis thaliana, for example, and are made available by differentculture collections (“Stock centre”, e.g. Salk Institute GenomicAnalysis Laboratory, 10010 N. Torrey Pines Road, La Jolla, Calif. 92037,http://signal.salk.edu/).

T-DNA mutagenesis is basically suitable for the production of the plantcells and plants according to the invention, which have a decreasedactivity of a cyclin SDS like protein.

In conjunction with the present invention, the term “foreign nucleicacid molecule” is understood to mean such a nucleic acid molecule thateither does not occur naturally in the corresponding wild type plantcells or plants, or that does not occur naturally in the concretespatial arrangement in wild type plant cells or plants, or that islocalised at a place in the genome of the plant cell or plant at whichit does not occur naturally. Preferably, the foreign nucleic acidmolecule is a recombinant molecule, which consists of differentelements, the combination or specific spatial arrangement of which doesnot occur naturally in plant cells or plants.

In principle, a foreign nucleic acid molecule can be any nucleic acidmolecule that effects a decrease in the activity of a cyclin SDS likeprotein. Such kind of nucleic acid molecules have been described hereinabove.

In conjunction with the present invention, the term “genome” is to beunderstood to mean the totality of the genetic material present in aplant cell. It is known to the person skilled in the art that, as wellas the cell nucleus, other compartments (e.g. plastids, mitochondriona)also contain genetic material.

A large number of techniques are available for the introduction of DNAinto a plant host cell. These techniques include the transformation ofplant cells with T-DNA using Agrobacterium tumefaciens or Agrobacteriumrhizogenes as the transformation medium, the fusion of protoplasts,injection of nucleic acids, the electroporation of nucleic acids, theintroduction of nucleic acids by means of the biolistic approach as wellas other possibilities.

The use of Agrobacteria-mediated transformation of plant cells has beenintensively investigated and adequately described in EP 120516; Hoekema,I N: The Binary Plant Vector System Offsetdrukkerij Kanters B. V.,Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4,1-46 and by An et al. EMBO J. 4, (1985), 277-287. For the transformationof potato, see Rocha-Sosa et al., EMBO J. 8, (1989), 29-33, for example.

The transformation of monocotyledonous plants by means of vectors basedon Agrobacterium transformation has also been described (Chan et al.,Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994)271-282; Deng et al, Science in China 33, (1990), 28-34; Wilmink et al.,Plant Cell Reports 11, (1992), 76-80; May et al., Bio/Technology 13,(1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992),550-555; Ritchie et al, Transgenic Res. 2, (1993), 252-265). Analternative system to the transformation of monocotyledonous plants istransformation by means of the biolistic approach (Wan and Lemaux, PlantPhysiol. 104, (1994), 37-48; Vasil et al., Bio/Technology 11 (1993),1553-1558; Ritala et al., Plant Mol. Biol. 24, (1994), 317-325; Spenceret al., Theor. Appl. Genet. 79, (1990), 625-631), protoplasttransformation, electroporation of partially permeabilised cells and theintroduction of DNA by means of glass fibres. In particular, thetransformation of maize has been described in the literature many times(cf. e.g. WO95/06128, EP0513849, EP0465875, EP0292435; Fromm et al.,Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2,(1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Morocet al., Theor. Appl. Genet. 80, (1990), 721-726). The successfultransformation of other types of cereal has also already been described,for example for barley (Wan and Lemaux, see above; Ritala et al., seeabove; Krens et al., Nature 296, (1982), 72-74) and for wheat (Nehra etal., Plant J. 5, (1994), 285-297; Becker et al., 1994, Plant Journal 5,299-307). All the above methods are suitable within the framework of thepresent invention. Transformation of vegetable crops is also commonlyknown in the art. In Curtis (2012, Springer Science & Business Media,ISBN: 1402023332, 9781402023330) besides others, methods for thetransformation of coffee, pineapple, pear, radish, carrot, pea, cabbage,cauliflower and watermelon is disclosed. Transformation of vegetablecrops like banana, citrus, mango, papaya, watermelon, avocado, grape,(sweet) melon, kiwifruit, coffee, cacao have been described in Pua andDavey (2007, Springer Science & Business Media, ISBN: 3540491619,9783540491613).

For expressing nucleic acid molecules, like those conferring a genesilencing effect or being used for introducing mutations into an allelein plant cells or plants, these nucleic acid are preferably linked withregulatory DNA sequences, including those which initiate transcriptionin plant cells (promoters). At the same time, the promoter can be chosenso that expression takes place constitutively or only in a certaintissue, at a certain stage of the plant development or at a timedetermined by external influences. The promoter can be homologous orheterologous both with respect to the plant and with respect to thenucleic acid molecule. The nucleic acid molecules therefore commonly arenot naturally occurring in plants but are recombinant nucleic acidmolecules, meaning that the combination of different genetic elements(e.g. coding sequences, RNAi complementary sequence, promoters)comprised by the nucleic acid molecule are not present in thiscombination in nature.

Suitable promoters are commonly known in the art, for example, thepromoter of the 35S RNA of the cauliflower mosaic virus and theubiquitin promoter from maize for constitutive expression, the patatinpromoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) fortuber-specific expression in potatoes or a promoter, which only ensuresexpression in photosynthetically active tissues, e.g. the ST-LS1promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987),7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or, forendosperm-specific expression of the HMG promoter from wheat, the USPpromoter, the phaseolin promoter, promoters of zein genes from maize(Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., PlantMol. Biol. 15 (1990), 81-93), glutelin promoter (Leisy et al., PlantMol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366;Yoshihara et al., FEBS Lett. 383 (1996), 213-218) or shrunken-1 promoter(Werr et al., EMBO J. 4 (1985), 1373-1380). However, promoters can alsobe used, which are only activated at a time determined by externalinfluences (see for example WO 9307279). Promoters of heat-shockproteins, which allow simple induction, can be of particular interesthere. Furthermore, seed-specific promoters can be used, such as the USPpromoter from Vicia faba, which guarantees seed-specific expression inVicia faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993),669-679; Bäumlein et al., Mol. Gen. Genet. 225 (1991), 459-467).

The recombinant nucleic acid molecule may also contain a terminationsequence (polyadenylation signal), which is used for adding a poly-Atail to the transcript. A function in the stabilisation of thetranscripts is ascribed to the poly-A tail. Elements of this type aredescribed in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29)and can be exchanged at will.

Intron sequences can also be present, e.g. between the promoter and thecoding region. Such intron sequences can lead to stability of expressionand to increased expression in plants (Callis et al., 1987, Genes Devel.1, 1183-1200; Luehrsen, and Walbot, 1991, Mol. Gen. Genet. 225, 81-93;Rethmeier, et al., 1997; Plant Journal. 12(4): 895-899; Rose andBeliakoff, 2000, Plant Physiol. 122 (2), 535-542; Vasil et al., 1989,Plant Physiol. 91, 1575-1579; X U et al., 2003, Science in China SeriesC Vol. 46 No. 6, 561-569). Suitable intron sequences are, for example,the first intron of the sh1 gene from maize, the first intron of thepolyubiquitin gene 1 from maize, the first intron of the epsps gene fromrice or one of the two first introns of the PAT1 gene from Arabidopsis.

What has been described herein concerning the verification if a planthas a mutation in a nucleic acid sequence or a gene or allele encoding acyclin SDS like protein and for growing/cultivating the plants for themethod for producing a plant according to the invention is alsoapplicable here for the method comprising introducing a foreign nucleicacid molecule into a plant cell according to the invention.

In a further embodiment of the present invention the methods forproducing a plant according to the invention and the method according tothe invention comprising introducing a foreign nucleic acid moleculeinto a plant cell comprise a further step consisting of the productionof further plants from the plants obtained from step d) in each of themethods according to the invention. The further plants produced arecharacterised in that they comprise at least one mutant allele of acyclin SDS like protein encoding gene or that they have a decreasedactivity of a cyclin SDS like protein due to the introduction of aforeign nucleic acid molecule as described herein above. These furtherplants can be produced by means of vegetative (agamic) or generative(gamic, sexual) reproduction. Suitable for vegetative propagation are,for example, cuttings, in vitro tissue, cell, protoplast, embryo orcallus cultures, micropropagation, rhizomes or tubers. Other propagationmaterial includes, for example, fruits, seeds, seedlings, beingheterozygous or homozygous for a mutant allele of a cyclin SDS likeprotein encoding genes, etc.

Techniques for vegetative (agamic) propagation, includingmicropropagation of plants are well known in the art and e.g. describedfor banana, citrus, mango, papaya, avocado, (sweet) melon, have beendescribed in Pua and Davey (2007, Springer Science & Business Media,ISBN: 3540491619, 9783540491613). Sultana and Rhaman (2012, LAP LambertAcademic Publishing, ISBN-13: 978-3-8484-3937-9) e.g. disclose varioustissue culture and micropropagation methods for watermelon.

Plants obtainable/obtained by a method according to the inventioncomprising introducing a foreign nucleic acid molecule into a plantcell, are also an embodiment of the invention.

A further embodiment of the invention concerns a method for propagatingseedless fruit producing plants comprising the steps of

-   a) obtaining seeds from which a plant according to the invention    grows or obtaining seeds deposited under accession No. NCIMB 42532    or progeny thereof,-   b) growing plants from the seeds obtained in step a),-   c) selecting seedless fruit producing plants from the plants grown    under step b),-   d) propagating the plants selected under step c) by a method    selected from the group consisting of    -   i) grafting of parts of plants selected under step c) to another        rootstock,    -   ii) cultivating parts of plants selected under step c) in in        vitro tissue culture and optionally regenerating new plants from        the tissue culture,    -   iii) optionally producing embryo or callus cultures from parts        of plants selected under step c) and optionally regenerating new        plants from the tissue culture,    -   iv) optionally producing further plants by micropropagation        techniques.

Propagation of plant parts by grafting and propagation and optionallyregeneration of plants by tissue culture methods are well known in theart. Such methods are described in various scientific publications andare reviewed and summarized in a number of scientific books, like e.g.Smith (2012, Academic Press, ISBN-13: 978-0124159204), Gayatri &Kavyashree (2015, Alpha Science International Ltd, ISBN-13:978-1842659618) etc. For watermelon, respective methods are describede.g. by Sultana and Rhaman (2012, LAP Lambert Academic Publishing,ISBN-13: 978-3848439379).

Plants or plant parts obtainable/obtained by methods for propagatingseedless fruit producing plants according to the invention are also anembodiment of the invention.

In a further embodiment of the invention, all methods for producing aplant according to the invention or optionally the methods forpropagating seedless fruit producing plants according to the inventiondisclosed herein are used for producing a plant according to theinvention.

A further embodiment of the invention concerns propagation material ofplants according to the invention, and/or propagation material of plantscomprising plant cells according to the invention or propagationmaterial of plants obtainable/obtained by a method according to theinvention for production of a plant or propagation material of plantsobtainable/obtained by a method according to the invention comprisingintroducing a foreign nucleic acid molecule into a plant cell orpropagation material of plants obtainable/obtained from a plantoptionally obtainable by a method for propagating seedless fruitproducing plants according to the invention. A specific comprisedembodiment of the invention is propagation material of plantsobtainable/obtained from seeds deposited under accession number NCIMB42532, preferably propagation material of plants obtainable/obtainedfrom seeds deposited under accession number NCIMB 42532 beingheterozygous or homozygous for the mutant allele of a cyclin SDS likeprotein encoding gene. Also comprised by the invention is propagationmaterial of plants heterozygous or homozygous for a mutant allele of acyclin SDS like protein encoding gene, wherein the propagation materialis obtained/obtainable from plants originating from a crossing of aplant obtained from seeds of deposit accession number NCIMB 42532 withanother plant.

Here, the term “propagation material” comprises those components of theplant which are suitable for generating progeny via the vegetative(agamic) or generative (gamic, sexual) route. Suitable for vegetativepropagation are, for example, cuttings, in vitro tissue, cell,protoplast, embryo or callus cultures, micropropagation methods,rhizomes or tubers. Other propagation material includes, for example,fruits, seeds, seedling, being heterozygous for a mutant allele of acyclin SDS like protein encoding gene etc. The propagation material inone aspect takes the form of cuttings which are propagated, e.g. bygrafting to another rootstock, or in vitro tissue culture material, inparticular embryo cultures. In particular preferred is propagationmaterial in the form of in vitro tissue culture material, particularlyin vitro embryo cultures. A plant produced by vegetative propagation isherein also referred to as a vegetatively propagated plant. Especiallyplants in which the mutant cyclin SDS like allele is present inhomozygous form are preferably propagated vegetatively (as their fruitsare seedless).

A further embodiment of the invention concerns parts of plants accordingto the invention, and/or parts of plants comprising plant cellsaccording to the invention or parts of plants obtainable/obtained by amethod according to the invention for production of a plant or parts ofplants obtainable/obtained by a method according to the inventioncomprising introducing a foreign nucleic acid molecule into a plant cellor parts of plants obtainable/obtained from a plant optionallyobtainable by a method for propagating seedless fruit producing plantsaccording to the invention. A further comprised embodiment of theinvention concerns parts of plants obtainable/obtained from seedsdeposited under accession number NCIMB 42532 (or from progeny thereof),preferably plant parts of plants obtainable/obtained from seedsdeposited under accession number NCIMB 42532 (or from progeny thereof)being heterozygous or homozygous for the mutant allele of a cyclin SDSlike protein encoding gene. Also comprised by the invention are plantparts of plants heterozygous or homozygous for a mutant allele of acyclin SDS like protein encoding gene, wherein the plant parts areobtained/obtainable from plants originating from a crossing of a plantobtained from seeds of deposit accession number NCIMB 42532 with anotherplant.

A further embodiment of the invention concerns a method for productionof a seedless fruit comprising growing a plant according to theinvention and/or growing a plant comprising plant cells according to theinvention or growing a plant obtainable/obtained by a method forproduction of a plant according to the invention or growing fruits ofplants obtainable/obtained by a method according to the inventioncomprising introducing a foreign nucleic acid molecule into a plant cellor growing a plant obtainable/obtained from a plant optionallyobtained/obtainable by method for propagating seedless fruit producingplants according to the invention in a field or in a greenhouse (e.g. aglasshouse, tunnel or net-house), allowing the plant to be pollinatedand harvesting the seedless fruit. Preferably the plant grown in themethod for producing seedless fruits according to the invention ishomozygous for a mutant allele of a cyclin SDS like protein encodinggene.

It has been surprisingly found that pollination of plants comprising amutant allele of a cyclin SDS like protein encoding gene in homozygousstate with pollen from a different plant will result in the plant toproduce seedless fruits. It is not decisive if the stigma of a plantbeing homozygous for a mutant allele of a cyclin SDS like proteinencoding gene is pollinated by pollen comprising a mutant allele of acyclin SDS like protein encoding gene or by pollen comprising a wildtype allele of a cyclin SDS like protein encoding gene. In any case,independent of the genotype of the pollen, the female plant beinghomozygous for a mutant allele of a cyclin SDS like protein encodinggene will produce seedless fruits.

Even when cross-pollinated by pollen from wild type plants, plantsaccording to the invention being homozygous for a mutant allele of acyclin SDS like protein encoding gene will produce seedless fruits.Thus, when cultivating plants according to the invention, in any caseseedless fruits will be obtained.

“Greenhouse” shall be understood in connection with the presentinvention to mean a building or compartment used for growing plantswhich has a roof and walls of transparent material consisting of glass,plastic, polyethylene, gaze, netting or the like. Greenhouses may haveor not have further technical equipment for heating, cooling, shading,automatic watering, fertilisation, carbon dioxide concentrationadjustment etc. Greenhouses with any type of technical equipment shallbe comprised by the term “greenhouse” as used herein.

Another embodiment of the invention concerns fruits of orobtainable/obtained from plants according to the invention, and/orfruits comprising plant cells according to the invention or fruits ofplants obtainable/obtained by a method according to the invention forproduction of a plant or fruits of plants obtainable/obtained by amethod according to the invention comprising introducing a foreignnucleic acid molecule into a plant cell or fruits of plantsobtainable/obtained from a plant obtained/obtainable by a methodaccording to the invention for propagating seedless fruit producingplants or a fruit obtainable/obtained by a method according to theinvention for production of a seedless fruit. A further comprisedembodiment of the invention concerns fruits of plantsobtainable/obtained from seeds deposited under accession number NCIMB42532 (or progeny thereof), preferably fruits of plantsobtainable/obtained from seeds deposited under accession number NCIMB42532 (or progeny thereof) being heterozygous or homozygous for themutant allele of a cyclin SDS like protein encoding gene. Also comprisedby the invention are fruits heterozygous or homozygous for a mutantallele of a cyclin SDS like protein encoding gene, wherein the fruitsare obtained/obtainable from plants originating from a crossing of aplant obtained from seeds of deposit accession number NCIMB 42532 withanother plant. The fruits can be heterozygous for a mutant allele of acyclin SDS like protein encoding gene and produce seed bearing fruits orcan be homozygous for an allele of a cyclin SDS like protein encodinggene and produce seedless fruits. Fruits being heterozygous for anallele of a cyclin SDS like protein encoding gene can be used forpropagating plants comprising a mutant allele of a cyclin SDS likeprotein encoding gene. Preferably, the fruits according to the inventionare homozygous for a mutant allele of a cyclin SDS like protein encodinggene and/or produce seedless fruits. Seedless fruits for logical reasonsare not eligible for growing further plants from these fruits.Therefore, one embodiment of the invention concerns fruit according tothe invention which is a seedless fruit which is not eligible forpropagation or which cannot propagate or which is a non-propagating.

A further embodiment of the invention concerns the use of a nucleic acidmolecule encoding a cyclin SDS like protein selected from the groupconsisting of

-   a) Nucleic acid molecules, which encode a protein with the amino    acid sequence given under SEQ ID NO 2 or SEQ ID NO 4 or SEQ ID NO 6    or SEQ ID NO 12 or SEQ ID NO 19 or SEQ ID NO 20,-   b) Nucleic acid molecules, which encode a protein, the sequence of    which has an identity of at least 58% or at least 60%, preferably at    least 70%, more preferably at least 80%, even further preferred at    least 90% or particularly preferred at least 95% with the amino acid    sequence given under SEQ ID NO 2 or SEQ ID NO 4 or SEQ ID NO 6 or    SEQ ID NO 12 or SEQ ID NO 19 or SEQ ID NO 20;-   c) Nucleic acid molecules, which comprise the nucleotide sequence    shown under SEQ ID NO 1 or SEQ ID NO 3 or SEQ ID NO 5 or SEQ ID NO    17 or a complimentary sequence;-   d) Nucleic acid molecules, which have an identity of at least 58% or    at least 60%, preferably at least 70%, more preferably at least 80%,    even further preferred at least 90% or particularly preferred at    least 95% with the nucleic acid sequences described under c);-   e) Nucleic acid molecules, which hybridize with at least one strand    of the nucleic acid molecules described under a), b), c), or d)    under stringent conditions;-   f) Nucleic acid molecules, the nucleotide sequence of which deviates    from the sequence of the nucleic acid molecules identified under a)    or b) due to the degeneration of the genetic code; and-   g) Nucleic acid molecules, which represent fragments, allelic    variants and/or derivatives of the nucleic acid molecules identified    under a), b), c) or d)    for the production of a plant producing seedless fruits.

In a preferred embodiment of the use of a nucleic acid molecule encodinga cyclin SDS like protein, the nucleic acid molecule encoding a cyclinSDS like protein is used for the production of plants according to theinvention, in a particular preferred embodiment, the use of a nucleicacid molecule encoding a cyclin SDS like protein is used for productionof plants producing seedless fruits according to the invention.

In another preferred embodiments, a nucleic acid molecule encoding acyclin SDS like protein is used for the production of a plant partaccording to the invention or a fruit according to the invention.

In a further preferred embodiment a nucleic acid molecule encoding acyclin SDS like protein, is used in any of the methods of the inventiondisclosed herein. The nucleic acid molecules encoding a cyclin SDS likeprotein can e.g. be used in a method according to the invention forproduction of a plant or in a method according to the inventioncomprising introducing a foreign nucleic acid molecule into a plant cellor in a method according to the invention for propagating seedless fruitproducing plants according to the invention or in a method according tothe invention for production of a seedless fruit.

In another preferred embodiment, a nucleic acid molecule encoding acyclin SDS like protein is used for identifying if a plant cell or plantcomprises a mutant allele of a cyclin SDS like protein encoding gene orif a plant cell or plant synthesises a mutant mRNA encoding a cyclin SDSlike protein or if a plant cell or plant has a decreased activity of acyclin SDS like protein. Preferably the nucleic acid molecule encoding acyclin SDS like protein is used for identifying if a seedless fruitproducing plant comprises a mutant allele of a cyclin SDS like proteinencoding gene or if a plant synthesises a mutant mRNA encoding a cyclinSDS like protein or if a plant has a decreased activity of a cyclin SDSlike protein. How such plants can be identified has been describedherein above and is applicable hereto accordingly.

Preferred embodiments concerning nucleic acid molecules encoding cyclinSDS like proteins have been described herein above and are applicablefor the uses according to the invention accordingly.

In one aspect a screening method for identifying and/or selecting seeds,plants or plant parts or DNA from such seeds, plants or plant partscomprising in their genome a mutant allele of a cyclin SDS like proteinencoding gene is provided.

The method comprises screening at the DNA, RNA (or cDNA) or proteinlevel using known methods, in order to detect the presence of the mutantallele. There are many methods to detect the presence of a mutant alleleof a gene.

For example if there is a single nucleotide difference (singlenucleotide polymorphism, SNP) between the wild type and the mutantallele, a SNP genotyping assay can be used to detect whether a plant orplant part or cell comprises the wild type nucleotide or the mutantnucleotide in its genome. For example the SNP can easily be detectedusing a KASP-assay (see world wide web at kpbioscience.co.uk) or otherSNP genotyping assays. For developing a KASP-assay, for example 70 basepairs upstream and 70 base pairs downstream of the SNP can be selectedand two allele-specific forward primers and one allele specific reverseprimer can be designed. See e.g. Allen et al. 2011, Plant BiotechnologyJ. 9, 1086-1099, especially p 097-1098 for KASP-assay method.

Equally other genotyping assays can be used. For example, a TaqMan SNPgenotyping assay, a High Resolution Melting (HRM) assay, SNP-genotypingarrays (e.g. Fluidigm, Illumina, etc.) or DNA sequencing may equally beused.

Genotyping of diploid plants or plant parts (cells, leaves, DNA, etc.)can distinguish SNP genotypes, e.g. plants or parts comprising CC fornucleotide 1687 of SEQ ID NO: 1 (homozygous for the wild typenucleotide) can be distinguished from plants or parts comprising CT fornucleotide 1687 of SEQ ID NO: 1 (heterozygous for the mutant nucleotide)in their genome. Genotyping of tetraploid plants or plant parts (cells,leaves, DNA, etc.) can be done in the same way as for diploids, usingfor example a KASP-assay to distinguish SNP genotypes, e.g. plants orparts comprising CCCC for nucleotide 1687 of SEQ ID NO: 1 (homozygousfor the wild type nucleotide) can be distinguished from plants or partscomprising other genotypes for the SNP, e.g. CCCA, CCAA, etc. in theirgenome. The same applies for triploids. The same also applies for otherpolyploids.

In a preferred aspect the above methods, plants, plant cells and plantparts which comprise at least one copy of a mutant allele of a cyclinSDS like protein encoding gene, are watermelon plants, especiallycultivated watermelon, e.g. diploid, tetraploid or triploid cultivatedwatermelon.

The watermelon plants may be breeding lines or varieties. The mutantallele of a cyclin SDS like protein encoding gene may be generated in,or introduced into (e.g. from seeds deposited under NCIMB42532 orprogeny thereof), any cultivated watermelon to produce lines orvarieties comprising the mutant allele of the SDS like protein,preferably in homozygous form. Cultivated watermelons produce diversefruit sizes (e.g. very small, as described in WO2012069539, e.g. lessthan 0.9 kg or even equal to or less than 0.65 kg; personal-size ofabout 3-7 pounds, i.e. about 1.4 to 3.2 kg; icebox sizes of about 6-12pounds, i.e. about 2.7 to 5.5 kg; and larger sizes of up to 35 pounds,i.e. about 15.9 kg), fruit flesh colors, and fruit shapes and withdifferent rind colors. The mutant allele may, therefore, be introducedinto cultivated watermelon producing any fruit shape (e.g. elongate,oval, oval elongated, blocky, blocky elongated, spherical or round),fruit surface (e.g. furrow, smooth), flesh color (e.g. red, dark red,scarlet red, coral red, orange, salmon or pink, yellow, canary yellow orwhite), rind color (e.g. light green; dark green; green-striped withnarrow, medium or wide stripes; grey types; with or without spotting;Golden yellow; Crimson type rind, Jubilee type rind; Allsweet type rind;black/dark green), rind thickness, rind toughness, rind pattern (e.g.striped, non-striped, netted), flesh structure/flesh firmness, lycopeneand/or vitamin content, different sugar to acid ratios, very good fruitflavour, etc. by breeding. See Guner and Wehner 2004, Hort Science39(6): 1175-1182, in particular pages 1180-1181 describing genes forfruit characteristics. Generally important breeding objectives are earlymaturity, high fruit yield, high internal fruit quality (good uniformcolor, high sugar, proper sugar:acid ratio, good flavour, high vitaminand lycopene content, firm flesh texture, non-fibrous flesh texture,freedom from defects such as hollow heart, rind necrosis, blossom-endrot or cross stitch and good rind characteristics andcracking-resistance). The fruits produced by the line or variety arepreferably marketable fruits. In one aspect the average brix is at least6.0, 7.0, 8.0 or at least 9.0, preferably at least 10.0, more preferablyat least 11.0 or more.

Fruit color may be any color, such as red, dark red, scarlet red, coralred, orange, salmon, pink, pinkish red, yellow, canary yellow or white.Preferably the fruit flesh color is uniform.

DEPOSIT INFORMATION

Diploid Citrullus lanatus seeds of plants segregating for a mutantallele of a cyclin SDS like protein encoding gene have been deposited byNunhems B. V. under the Budapest Treaty under accession No. NCIMB 42532at the NCIMB Ltd., Bucksburn Aberdeen AB21 9YA, Scotland on 27 Jan.2016. For the seed deposit the allele of the cyclin SDS like proteinencoding gene was designated emb1. The Expert solution applies.

The deposited seeds were obtained from a self-pollinted back-cross of aplant homozygous for the emb1 mutant allele with plants homozygous forthe emb1 wild type allele. Therefore 25% of the deposited seeds arehomozygous for the emb1 mutant allele and produce seedless fruits, 50%are heterozygous for the mutant allele and 25% are homozygous for thewild type allele, encoding the wild type cyclin SDS like protein.

Access to the deposits will be available during the pendency of thisapplication to persons determined by the Commissioner of Patent andTrademarks to be entitled thereto upon request.

Subject to 37 C.F.R. § 1.808(b), all restrictions imposed by thedepositor on the availability to the public of one or more deposits willbe irrevocably removed upon the granting of the patent by affordingaccess to the deposits. The deposits will be maintained for a period of30 years, or 5 years after the most recent request, or for theenforceable life of the patent whichever is longer, and will be replacedif it ever becomes nonviable during that period. Applicant does notwaive any rights granted under this patent on this application or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

Sequence Description

-   SEQ ID NO 1: Genomic sequence of a wild type cyclin SDS like protein    encoding gene from Citrullus lanatus.-   SEQ ID NO 2: Amino acid sequence of a SDS like protein from    Citrullus lanatus. The amino acid sequence is derivable from the    coding sequence of SEQ ID NO 1.-   SEQ ID NO 3: mRNA sequence of a mutant allele of a cyclin SDS like    protein from Citrullus lanatus.-   SEQ ID NO 4: Amino acid sequence of the mutant allele of a SDS like    protein. The amino acid sequence is derivable from SEQ ID NO 3.-   SEQ ID NO 5: Nucleic acid sequence of a wild type cyclin SDS like    protein encoding gene from Cucumis melo.-   SEQ ID NO 6: Amino acid sequence of a SDS like protein from Cucumis    melo. The amino acid sequence is derivable from the coding sequence    of SEQ ID NO 5.-   SEQ ID NO 7: Artificial sequence used as primer (A4532) in PCR    and\or sequencing reactions.-   SEQ ID NO 8: Artificial sequence used as primer (A4533) in PCR    and\or sequencing reactions.-   SEQ ID NO 9: Artificial sequence used as primer (A4534) in PCR    and\or sequencing reactions.-   SEQ ID NO 10: Artificial sequence used as primer (A4535) in PCR    and\or sequencing reactions.-   SEQ ID NO 11: Artificial sequence used as primer (A4538) in PCR    and\or sequencing reactions.-   SEQ ID NO 12: Amino acid sequence of a SDS like protein from Cucumis    sativus.-   SEQ ID NO 13: sequence of sample 114 and 115 of FIG. 2-   SEQ ID NO 14: sequence of sample 114 and 115 of FIG. 2-   SEQ ID NO 15: sequence of sample 116 and 117 of FIG. 2-   SEQ ID NO 16: sequence of sample 116 and 117 of FIG. 2-   SEQ ID NO 17: cDNA sequence of a watermelon mutant cyclin SDS like    gene comprising a C to T mutation at nucleotide 670 resulting in a    stop codon at nucleotide 670 to 671-   SEQ ID NO 18: watermelon mutant cyclin SDS like protein encoded by    the cDNA of SEQ ID NO 17-   SEQ ID NO 19: Amino acid sequence of a wild type SDS like protein    from Solanum lycopersicum-   SEQ ID NO 20: Amino acid sequence of a wild type SDS like protein    from Capsicum annuum

DESCRIPTION OF THE FIGURES

FIG. 1 Watermelon fruits from wild type plants (1A), wild type plantscompared to fruits from EMB1 mutant plants (1B), slice of seedless fruitfrom an EMB1 mutant plant (1C) and opened embryoless seed of an EMB1mutant plant (1D).

FIG. 2 Sequence alignment comparing the sequences obtained for SampleNumbers 114, 115, 116 and 117. The number in the top right sideindicates the nucleotide position in the corresponding sequence shownunder SEQ NO 1. Sample Numbers 116 and 117 (SEQ ID. 15, SEQ. ID 16) areobtained from EMB1 mutant plants. Sample Numbers 114 and 115 (SEQ. ID.13, SEQ. ID 14) are obtained from wild type plants.

FIG. 3 Electrophoretic analysis of the PCR products obtained from cDNAof Sample Numbers 114, 115, 116 and 117 on a polyacrylamide gel. SampleNumbers 114 and 115 are obtained from wild type plants. Sample Numbers116 and 117 are obtained from EMB1 mutant plants.

GENERAL METHODS

1. Isolation of RNA

Young ovary tissue was cut into small pieces, frozen in liquid nitrogenand stored at −80° C. until further use. The frozen tissue pieces wereground into powder with piston and mortar in liquid nitrogen to keep thepowder frozen. 100 mg of the powder was used to isolate total RNA usinga plant RNA isolation kit according to the manufacturer's protocol(RNeasy Plant Mini Kit, Qiagen).

2. Preparation of cDNA

The RNA was treated with DNase (TURBODNA-free, Ambion) and 0.9 μg RNAwas used for reverse transcription according to the manufacturer'sprotocols (iScript cDNA Synthesiskit, BioRad).

3. PCR on cDNA

PCR took place in a total volume of 20 μl of buffer (Phire reactionbuffer, Thermo Fisher Scientific) containing 0.2 mM dNTP's, 0.4 μl PhireHot Start II DNA Polymerase (Thermo Fisher Scientific), 0.2504 of eachprimer and 0.4 μl cDNA mix. After initial denaturation step of 30minutes at 90° C., 40 cycles of 10 seconds at 98° C., 15 seconds at 60°C. and 30 seconds at 72° C. were performed and the reaction wasfinalized with 3 minutes at 72° C.

4. Sequencing

The PCR product size was analysed using QIAxcel Advanced System (Qiagen)and the PCR reaction mixture was sent to a service provider to besequenced (BaseClear, NL).

EXAMPLES

1. Isolation of Seedless Fruit Mutant

A mutant population was established by treating approximately 10.000watermelon seeds from an inbred line (WMZD0048TYY, abbreviated TYY inthe following) with EMS several hours and subsequently washing the seedsin streaming tap water for 30 minutes. After that the seeds were keptwet until sowing in soil. M1 Plants were grown from the mutagenizedseeds, self-pollinated and the seeds (M2 generation) were harvested.Eight seeds from each of 3000 M2 families were sown grown and mutantplants producing seedless fruits were isolated. One of these mutantplants was designated EMB1. Propagation of the EMB1 mutant plant wasperformed by grafting cuttings of the EMB1 mutant plant to rootstock ofa non-mutagenized watermelon plants.

2. Confirmation of Seedless Fruit Phenotype

The EMB1 mutant was back-crossed with the original non-mutagenizedwatermelon TYY inbred line, using pollen from the EMB1 mutant (BC1generation). 25% of the plants grown from the self-pollinated BC1generation did produce seedless fruits.

Pollen from the EMB1 mutant was also used for crossing with differentwatermelon inbred lines for establishing a mapping population. 25% ofself-pollinated plants of the mapping population produced seedlessfruits.

The results from the respective back-crosses and crosses wherein pollenfrom the EMB1 mutant was used to fertilise other inbred lines clearlydemonstrate that pollen of the EMB1 mutant is fertile.

In further crosses EMB1 mutant plants, homozygous for the emb1 mutantallele were used as female parent and pollinated with pollen fromvarious different other lines. 100% of plants from each of these crossesproduced seedless fruits.

Results obtained from the different crossings show that the emb1mutation is due to a single recessive allele. The results alsodemonstrate that the seedless fruit phenotype is maintained when pollenform seed producing plants is used to fertilise EMB1 mutant plants. Theseedless fruit phenotype therefore is not due to aberrant pollen of theEMB1 mutant but can be assigned to defects in embryo development.

3. Identification of the Gene Causing the Seedless Fruit Phenotype

The mapping population established by pollinizing different watermeloninbred lines with pollen from the EMB1 mutant plant was analysed and asingle nuclear polymorphism (SNP) was detected in the genomic sequenceshown under SEQ ID NO 1. SEQ ID NO 1 shows the sequence of the wild typeallele. In the respective allele of the EMB1 mutant plant the nucleotideguanine (G) at position number 2185 in SEQ ID NO 1 is replaced byadenine (A).

4. Analysis of mRNA Transcribed from emb1 Alleles

Flower buds of different size were harvested from field grown plants.Sample Numbers 114 and 115 are flower buds from plants of the originalinbred line, designated TYY, which was used for mutagenesis. TYY thusrepresent wild type plants comprising wild type emb1 alleles. SampleNumbers 115 and 116 are flower buds from seedless fruit producing EMB1mutant plants comprising mutant emb1 alleles. An overview of phenotypes,material harvested and analysed for respective Sample Numbers is givenin Table 1.

TABLE 1 Sample Plant Sample Number Name Tissue collected Phenotype 114TYY (101) Flower bud 1 mm Wild type seed bearing fruit 115 TYY (102)Flower bud 3-4 mm Wild type seed bearing fruit 116 EMB1 (103) Flower bud1 mm Seedless fruit 117 EMB1 (104) Flower bud 3-4 mm Seedless fruit

RNA was isolated from the flower buds of the different Sample numbers.700 ng RNA for each of the Sample numbers was used for cDNA synthesis. APCR reaction using primers A4532 (SEQ ID NO 7) and A4533 (SEQ ID NO 8)was performed on each of the cDNA samples obtained. The primers weredesigned to amplify a part of the emb1 allele of the coding sequenceindicated in SEQ ID NO 1. The PCR products were analysed on apolyacrylamide gel which is shown in FIG. 3 . From FIG. 3 it is clearlyderivable, that Sample numbers 116 and 117 did result a shorter PCRfragment than all the other Sample numbers. This clearly indicated thatthe mRNA of the emb1 mutant allele comprises a deletion of nucleotidescompared to the corresponding wild type allele in Sample numbers 114 and115.

5. cDNA Sequence Analysis of emb1 Alleles

cDNA of Sample Numbers 114, 115, 116 and 117 were sequenced using primerA4532 shown under SEQ ID NO 7, primer A4538 shown under SEQ ID NO 11,primer A4534 SEQ ID NO 9 and primer A4535 SEQ ID NO 10. The sequencesobtained from each of Sample Numbers 116 and 117 is shown under SEQ IDNO 3 as mRNA molecule. The sequences obtained from each of SampleNumbers 114 and 115 is identical to the coding sequence indicated in SEQID NO 1. Comparison of the sequences obtained from Sample Numbers 114,115, 116 and 117 showed that the sequences of each of Sample Number 116and 117 has a deletion of 16 consecutive nucleotides compared to thesequences of each of Sample Number 114 and 115. In addition, sequencesof each of Sample Number 116 and 117 have a frame shift which causes apre-mature stop codon in the coding sequence, compared to the sequencesof each of Sample Number 114 and 115. An alignment of the sequence partsconcerned is shown in FIG. 2 .

It is concluded, that the emb1 mutant allele of Sample Numbers 116 and117 is transcribed into an mRNA which has a deletion, a frame shift inthe reading frame and a pre-mature stop codon compared to mRNAtranscribed from the wild type allele of Sample Numbers 114 and 115. Inaddition, it can be seen from FIG. 2 that the mRNA transcribed from theemb1 mutant allele of Sample Numbers 116 and 117 encodes a proteinwherein 8 amino acids are exchanged compared to protein encoded by themRNA transcribed from the wild type allele of Sample Numbers 114 and115.

6. Generation of Another Watermelon Mutant Plant

Knowing the cyclin SDS like gene sequence made it possible to generateother mutant alleles in the SDS like gene. The EMS mutagenized TILLINGpopulation was screened with primers designed on a domain in which EMSmutations could convert an amino acid encoding codon into a stop codon.

Forward primer: (SEQ ID NO: 21) CGAAGAGAAAGGATTAGACGTTG Reverse primer:(SEQ ID NO: 22) TCTGAGCAGTCAGTATCAGACG

A plant was identified comprising a mutant cyclin SDS like allele. Theidentified allele comprises a single nucleotide replacement atnucleotide 1687 of SEQ ID NO: 1, leading to a stop codon. The mutantallele thus encodes a truncated cyclin SDS like protein comprising onlyamino acids 1 to 223 of the wild type protein of SEQ ID NO: 2. The cDNAof the mutant allele is provided in SEQ ID NO: 17 and the truncatedprotein encoded by the mutant allele is provided in SEQ ID NO: 18.

The invention claimed is:
 1. A watermelon plant or seed comprising amutant allele of a gene encoding a cyclin SDS like protein, wherein themutant allele comprises a mutation in the promoter sequence resulting inno gene expression compared to a corresponding wild type allele orwherein the mutant allele encodes a non-functional protein comprising adeletion, truncation, insertion or replacement of one or more aminoacids, compared to the protein encoded by the wild type allele, whereinthe cyclin SDS like protein of the wild type allele is encoded by one ofthe following nucleic acid molecules: a) a nucleic acid molecule, whichencodes a protein with the amino acid sequence given under SEQ ID NO: 2;b) a nucleic acid molecule, which encodes a protein, the sequence ofwhich has an identity of at least 80% with the amino acid sequence givenunder SEQ ID NO: 2; c) a nucleic acid molecule, which comprises thenucleotide sequence shown under SEQ ID NO: 1 or the complimentarysequence thereof; or d) a nucleic acid molecule, which has an identityof at least 90% with one of the nucleic acid sequences described underc.
 2. The watermelon plant or seed of claim 1, wherein the mutant alleleof a gene encoding a cyclin SDS like protein encodes a non-functionalprotein in which one or more amino acids are inserted, replaced ordeleted in the conserved Cyclin N domain and/or Cyclin C domain of theprotein.
 3. A cell of a watermelon plant of claim
 1. 4. A seed fromwhich a watermelon plant of claim 1 can be grown.
 5. Parts of awatermelon plant of claim
 1. 6. Propagation material comprisingwatermelon plant cells of claim
 3. 7. A fruit comprising a watermelonplant cell of claim
 3. 8. The watermelon plant or seed of claim 1,wherein said plant is homozygous for the mutant allele of a geneencoding a cyclin SDS like protein.
 9. A method for production of aseedless fruit comprising growing a plant or seed of claim 8, allowingpollination of said plant, and harvesting the seedless fruit.
 10. Thepropagation material of claim 6, wherein the propagation material is avegetatively propagated plant.
 11. The watermelon plant or seed of claim1, wherein the mutant allele encodes a non-functional protein which istruncated and lacks the Cyclin_N and Cyclin_C domains.
 12. Thewatermelon plant or seed of claim 1, wherein the mutant allele encodes anon-functional protein which lacks amino acids encoded by exons 2, 3and/or 4 of the wild type cyclin SDS like protein.
 13. The watermelonplant or seed of claim 1, wherein the mutant allele comprises a mutationwhich results in a premature stop codon.
 14. The watermelon plant orseed of claim 1, wherein the cyclin SDS like protein of the wild typeallele is encoded by a nucleic acid molecule, which encodes a proteinwith the amino acid sequence given under SEQ ID NO:
 2. 15. Thewatermelon plant or seed of claim 1, wherein the cyclin SDS like proteinof the wild type allele is encoded by a nucleic acid molecule, whichencodes a protein, the sequence of which has an identity of at least 80%with the amino acid sequence given under SEQ ID NO:
 2. 16. Thewatermelon plant or seed of claim 1, wherein the cyclin SDS like proteinof the wild type allele is encoded by a nucleic acid molecule, whichcomprises the nucleotide sequence shown under SEQ ID NO: 1 or thecomplimentary sequence thereof.
 17. The watermelon plant or seed ofclaim 1, wherein the cyclin SDS like protein of the wild type allele isencoded by a nucleic acid molecule, which has an identity of at least90% with one of the nucleic acid sequences described under c).